Intraceiiular : Relationship to Osteogenic Differentiation and Cell Migration

Ron Zohar

A thesis submitted in conformity with the requirements For the Degree of Doctor of Philosophy, Graduate Department of Dentistry, University of Toronto

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David and Rachel Zohar ABSTRACT Osteopontin (OPN), a major component of the bone rnatrix, is expresseci at different stages of bone ce11 development To determine possible relationships between OPN expression and stages of osteogenic ce11 differentiation 1 perfomed analyses of intracellular OPN in early (proiiferating), sub-confluent (differentiating) and mature (mineralizing) cultures of fetal rat calvarial cells (FRCC7s)using a combination of flow cytometry and confocai microscopy. At each culture stage a high proportion (60 - 98%) of celts were immunoreactive for OPN (OPN+ve) which in combination with rnorphological characteristics can be used to analyze osteogenic differentiation. Within the OPN-ve cells a population of srnall cens with low cytoplasmic gfanulariw (S-celis) were isolated by flow cytometry and found to be enricheci in stem celis. Thus, fkeshly-sorted suspensions of irnmunostained S cells did not express differentiation-associated markers such as type 1, II, and III collagens, alkaline phosphatase or osteopontin. However, S celis expressed these markers together with OPN and CD44 following attachment. Upon plating, S celis also exhibited hi& proliferation capacity and in culture generated chondrocyte, adipocyte, and smooth muscle phenotypes, in addition to osteogenic cells. Limiting dilution analysis revealed a 3-fold enrichment of osteo-progenitors in the S cell population, which was the only population that demonstrated extensive self-renewal of cells with osteogenic potential. In OPN+ve cells, two patterns of intracellular immunostaining were observed; perinuclear, typical of secreted proteins, and perimernbranous. The perimembranous staining predominated in rnigrating FRCCs and fetd dermal fibroblasts, which contained higher levels of OPN than non-migrating cells. Using confocal microscopy and double- immunofluorescent labelhg the perimembranous OPN was found to CO-localizewith cell- surface CD44 and ezrin-radwn-rnoesin (ERM) protek. Moreover, the OPN was immunoprecipitated by CD44 antibodies as a complex with ERM proteins and CD44. OPN-/-fetai fibroblasts exhibited impaired abiiity to migrate and to attach to hyaluronan coated-beads. Co-localkation of OPN and CD44 was also demonstrated in activated human and in a metastatic epithelial tumor ce11 he. Collectively, these studies of intracellular OPN have identified stromd precursor cells with stem ceil characteristics and have revealed an intracellular form of OPN that appears to be a hctional component of the CD44-ERM ceii attachent cornplex in rnigrating cells. .. 11 The identification of stromal stem cells is a widely pumied dream of many scientists and 'liealers" of connective tissues. In the last 3 years under the guidance of my supervisor Dr. Jaro Sodek and the efforts of Dr. Chris McCulloch 1was fortunate to be part of a step towards the characterizaiion of mesenchymal stem cell. I'm gratefid to Dr. Sodek for his help and support during the 1st3 years and feel privileged to have been his graduate student- My appreciation is also extended to Dr. McCulloch for his intense involvernent in this project enabling an exciting multidiciplinary approach to be taken. I'm gratefûi to them especialiy for the love of science they have projected. 1 thank the M.R.C. Group in Periodontal Physiology, the Harron Trust Foundation and B. Curty Foundation and Alpha Omega Fraternity for their support. 1 thank the Faculty of Denb'stry for their support and hospitality in the Penodontal Department and also in Mount-Sinai Hospital, especially to Dr. H. Tenenbaum, Dr. R. Ellen, Dr. D. Mock and h. S. Golden and his fdly. 1 thank Sela Cheifetz for her advice and help. 1 also thank Harry Moe, Wilson Lee, Pam Arora and Kam-Ling Yao for their help in rny work. Special thanks are due to Elisa Krissilas who did far and above her office duties in helping me. Special thanks to Dr. Haim Ta1 who walked me through this joumey to Toronto

[ also thank Dr. Moses and Dr. Pitaru for their support. Also suicere thanks to my dear fiend Dr. Aaron Levy. Above al1 1thank My son Raz and my parents for their love and support. EDUCATION, AWARDS and PUBLICATIONS

1984-87 B. Med. Sc., Ben Gurion University Medical School 1987 DMD., School of Dental Medicine, Tel-Aviv University. 1991-94 Postgraduate program in Periodontics, Tel-Aviv University. 1994 Graduate program M.RC Group in Periodontal Physiology, Faculty of Dentistry, University of Toronto.

Awards and Fellowsk@s:

1994 - Medical Research Council Group Grant (Graduate student stipend) 1994 - B. Cürty Foundation, Fellowship, SwitzerIancL 1995 - Alpha ûmega Foundation, Fellowship. 1996 - Wilson G. Harron, Award and Fellowship, University of Toronto. 1997 - Graduate Research Day Second Place Award, Faculty of Dentistry, University of Toronto.

Publications:

1. Zohnr, R, Metzger, Z., Sukman, Y., and Gordon, M. (1986) Preventive Dentistry in Elementary schools, Modalities in community education. Tel - Aviv University Press.

2. Zohar, R, Metzger, Z., and Pitaru, S. (1988) Thesis : The effect of bacterial endotoxin-activated macrophages on fibroblast repopulation of an in vitro model. DMD thesis.

3. Zohar, R, Lee W., Arora P., Cheifetz S., McCulIoch, C.A.G., and Sodek, J. (1997) Single Ce11 Analysis of htracellular Osteopontin in Osteogenic Cultures of Fetal Rat Calvarial Cells. J. Cell. Physiol., l70:88-lOO.

4. Artzi, Z., Zohar, R, and Tal, H. (1997) Periodontal and peri-implant bone regeneration: Ciinical and histologic observations. Int. J. Periodont. Rest. Dent., 17(1): 63-72.

5. Sukhu, B., Rotenberg, B., Binkert, C., Kohno, H., Zohar, R, McCulloch, C.A.G. and Tenenbaum, H.C. (1997) Tamoxifen attenuates glucocorticoid actions on bone formation in vitro. Endocrinology, 138:3269-75.

6. Zohar, R, Sodek, J., and McCulloch, C.A.G. (1997). Characterizaion of stroma1 stem cells enriched by flow cytometry. Blood, 90:3471-8 1.

7. Zohar, R, Cheifetz S., McCulloch, C.A.G., and Sodek, J. (1997). Analysis of Intracelluiar Osteopontin as a Marker of Osteoblastic Ce11 Differentiation and Mesenchymal Cell Migration. Eur. J. Oral Biol., In press. 8. Zohar, R., McCulloch, CA-G., and Sodek J. (1998) Flow Cytometric Analysis of rhOP-1 (BMP-7) Responsive Subpopdations fkom Fetal Rat Calvaria Based on Intracellular Osteopontin Content. MWBiology, In press.

1. Zohar, R*, McCulloch, C.A.G., Sampath, TX, and Sodek, 1. 1996. Differentiation- stage-dependent modulation of OPN in fetal bone ceils by osteogenic protein-1(OP-1, BMP-7). Annual Meeting of the International Association of Dental Research, San Francisco, CA-, J. DENT.RES. 75:ABS. # 2814.

2. Zahar, R*, McCulloch, C-AG., and Sodek, L 1996. Single ceil analysis of discrete subpopulations of osteoponth positive and negative ceiis in rat osteogenic cultures. Annuai Meeting of the Clinical Research Society of Toronto, Ontario, ABS. #12.

3. Zohar, R*,McCulloch, C.A.G., and Sodek, J. 1996. Single cell andysis of discrete subpopulations of osteopontin positive and negative ceils in rat osteogenic cultures. Canadian Comective Tissue Conference, Toronto, Ontario.

4. Zohar, R*, McCulloch, C.A.G., and Sodek, J. 1997. Stromal stem celi fkom fetal rat calvaria: Isolation and characterization. Annual Meeting of the Intemational Association of Dental Research, Orlando, FL., J. DENT.RES. 76:ABS. # 1248,1997.

5. Zohar, R*, Moe, H., McCulloch, C.A.G., and Sodek, J. 1997. Stromal stem ceil fÏom fetal rat calvaria: Isolation and characterization. The Second Symposium on Skeletal Biology, London, Ontario, ABS. #18. TABLE OF CONTENTS

ABSTRACT ACKNOWLEDGEMENTS EDUCATION, AWARDS AND PUBLICATIONS TABLE OF CONTENTS LIST OF FTGWS LIST OF ABBREVLATIONS CHAPTER 1.-Review of The Literature Bone Development A. Characteristics of Bone as an Organ B. Bone Structure and ûrganization C. Bone Growth and Development D. Molecular Components of the Bone Matrix Bone Lheage Cultures A. Models in the Study of Bone Development 1. in vivo Modeis 2. lir vitro Models a) Tissue cultures b) himary ce11 cultures

C) Ce11 lines and transfarmed cells B. Developmental Stages in Primary Bone Ce11 Cultures C. Bone Matrix Proteins and Culture Maturation Bone Lineage Cells A. Definitions and Stages B. Early Precursors Cells of Bone C. Methods for Characterizing Early Precursors in Bone Lineage Ce11 Populations Ost eopontin

vi A- Structure 1. Characteristics of the Protein B. Regulation of OPN Expression 1. Homonal and Conml of OPN Synthesis 2. The OPN Gene and Transcriptional Regulation 3. Alternate Spliced Forms of OPN mRNA C. Tissue Distribution and Proposed Biological Roles D. OPN Signals through Entegrins and CD44 1. Integrins 2. CD44 V Statement of the Problem CHAPTER II- Single Ce11 Analysis of Fntracellular Osteopontin in Osteogenic Cultures of Fetal Rat Calvarid CeIls CHAPTER III- Characterization of Stroma1 Progenitor Cells Enriched by Flow Cytometry CHAPTER IV- Flow Cytometric Analysis of rhOP-1 (BMP-7) Responsive Subpopulations fkom Fetal Rat Calvaria Based on htracellular Osteopontin Content CHAPTER V-Intracellular OPN as an Integrai Part of a CD44-ERM Complex Involved in Ce11 Migration CHAPTER VI Discussion CHAPTER VII References

vii LIST OF FIGURES

Fig ïï. 1- Immunofluorescent staining of fetai rat calvarial ce& (FRCCs)with anti OPN antibodies Fig II. 2- Analysis of the relationship between cell number and intracelIular OPN fluorescence stalliing intensity in fetal rat calvarial ceils Fig II. 3- Relationship between cell numba OPN fluorescence and DNA content. Fig II. 4- Ceil cycle analysis of FRCCs stained for OPN and protein Fig II. 5- Bivariate plots of DNA content and protein content in FRCC Fig IL 6- Flow cytometry andysis of size & granularity of FRCCs Fig II. 7- Analysis of OPN in fetal and adult rat fibroblasts Fig II. & Analysis of OPN expression by flow cytometry, western blotting and RT-PCR. Fig II .9- Western blot of OPN protein in migrating FRCC's compared to non-rnigrating cells.

Fig m. 1- Sepration of FRCC by flow cytometry Fig III. 2-Analysis of ce11 proliferation and alkaline phosphatase expression. Fig III. 3-Limiting Dilution analyses of bone nodule-forming capacity using Iinear regression analysis Fig m. 4-Analysis of bone nodule formation. Fig III. 5-Evidence for pluripotentiality by formation of multiple ce11 types at clonal ce11 densities. Fig III. 6- Electron Microscopy of Sorted Cell Populations Table III. 1- Lmmunostaining of cells that were sorted and analyzed (Sorted-Cytospin) or sorted and plated overnight fiom S and L ce11 subpopulations. Table m. 2- Summary of the characterization of S and L ce11 subpopulations

viii Fig N. 1- CeU proliferation and aikaline phosphatase expression in FRCC and MC3T3-El cells in response to rhOP-1 at different stages of cellular differentiation. Fig W. 2- Bone nodule formation induced by rhOP-l in FRCCs. Fig IV. 3- Flow cytometric aualysis of the relationship between cell number and intracellular OPN in FRCCs. Fig IV. &Western blot analysis of OPN in FRCC extracts and conditioned medium. Fig W.5-Relationship between intracellular OPN and size and granularity of FRCCs. Fig W.6-Phaseîontrast rnicrographs of bone and cartilage colonies formed by S and L Cells. Fig IV. 7-Analysis of colony formation by S and L cells in response to rhOP- 1.

Fig V. 1- Analysis of biotinylated cell-surface proteins. Fig V. 2- Discrete immunocytochemical staining patterns for intracellular osteopontin Fig V. 3- Migration and binding of coated beads to OPN-/- and OPN+/+ embryonic mouse fibroblasts. Fig V. 4- Westem blot analysis of OPN and CD44 extracted fiom rat demal fibroblasts. Fig V. 5 - Analysis of biotinylated cell-surface proteins on Western blots. Fig V. 6- Localization of CD44 and OPN in relation to hyalumnan-coated beads. Fig V. 7- Westem blot analysis of whole ce11 and bead extracts from human periodontal ligament cells. Fig V. 8- Relationship of actin to the hyaluronan-bead cornplex. Fig V. 9- Confocal fluorescence microscopy of CD44, OPN and enin.

Fig VI. 1- Mode1 for the perimembranous intracellular OPN association. LIST OF ABBREVIATIONS

OPN Osteopontin Ca,, (Po,), (OH)Z Hydroxyapatite TPA 1 2-O-tetradecanoyl phorbol- 13-acetate FrPK Factor-independent protein kinase FRCCs Fetai rat calvaria cells BMP Bone morphogenetic proteins hop-1 Recombinant human osteogenic protein-1 ERM Ezrin, Radwn,Moesin RHAMM Receptor for hyaluronate-mediated motility ON SPARClosteonectin ROS 17/2.8 cells Rat osteusarcorna ceils EGF Epidermal AP Aikaline phosphatase a-MEM a-minimal essential medium PBS Phosphate bufTer saline rP Immunoprecipitation baer CHAPTER 1

REVIEW OF THE LITERATURE 1 Boue Development

A. Charocteristics of Bone as an ûrgun Bone is a specialized tissue that is formed by osteogenic cek which produce a unique mineraiized matrk. Bone is orgatuzed into a complex, multi-cellular-tissue cornplex that serves three major functions: (1) it provides rnechanicaI support through skeletai structures and aitachment for muscles and ligaments; (2) it protects intemal organs and the hematopoietic marrow; (3) it provides a metabolic reservoir for calcium and phosphate (Cormack, 1987; Murray, 1996). To meet these fùnctional requirements the mineralized matrix of bone tissue combines tende and compressive strength with a certain degree of flexibility. These propdes reflect the biophysical characteristics of the matrix, which comprises an extensive fhmework of collagen fibers irnpregnated with hydroxyapatite crystals and non-collagenous proteins associated with the collagen and mineral cornponents.

B. Bone Stnrcture and Organization Mature long bones and rnembranous bones have a highly mineraiized outer cortex that surrounds a loose cancellous bone. The cancellous bone forms a network of interconnected spicules of trabecular bone that encompass the marrow spaces. These spaces house the hematopoietic, fat and loose comective tissues. The surfaces of the compact cortical bone and the trabecular bone are covered by a layer of bone-fomiing cells or lining cells on the inner endosteal surfaces, which face the marrow spaces, or the outer periosteal surfaces. The growth, development and aging of bone involves remodeling mediated by cells in these investing layers. During development or repair newly-formed bone is of€en termed immature bone (Cormack, 1987). This bone is formed rapidly and is charactenzed by an apparently random network of collagen fibnls, which is the bais of the term "woven bone". Woven bone is more cellular than older bone and hydroxyapatite crystals are abundant between, as well as within, the collagen iibrils. It is also the type of bone formed by osteogenic tumors. Mature bone is forrned more slowly and in contrast to woven bone, the collagen fiber bdes are highiy ordered and fom alternathg laminated layers kom which the term ''Iameilar bone" is derived. In Iamellar bone the hydroxyapatite crystais are formed predominantly within the collagen fibrils producing a stronger bone which is found in the osteons of compact bone.

C. Bone Growth and Developmmt Osteoblasts are responsible for the production of the initial osteoid (ie. bone matrix) and also regulate its subsequent mineralization. Active osteoblasts, which are thought to originate fiom a stromal mesenchpal stem ce11 (Owen, 1985), are identified in tissue sections as a layer of cells arranged dong the newly forrned osteoid (Holtrop, 1975; Murray, 1996). Several layers of smdler cells that may include preosteoblastic cells cover the osteoblastic layer. Some osteoblasts become trapped in the matrix during mineralization; subsequently the ce11 body is reduced in size and develops long ce11 processes rich in microfilaments. This ce11 is an osteocyte and its processes are connected via gap junctions with other osteocytes and osteoblasts through a network of canaliculi in the mineralized matrix. The osteocyte is thought to play a role in bone turnover and the transfer of calcium ions nom the bone minera1 to the blood and tissue fluids. Two types of bone development can be identified histologically: intramembranous ossification occurs in the formation of the Bat bones of the skull and scapula, while endochondral ossification occurs commonly in long bones and vertebrae. In the development of intramembranous bone, stromal tissue cells condense and di fferentiate into osteoblasts, which synthesize the osteoid and reguiate its mineralization. in endochondral bone formation, stromal cells give rise to chondroblasts which secrete a cartilaginous matrix. This matrix is enriched with proteoglycans, which gives it a gel like consistency. Subsequent invasion of the cartilage matrix by blood vessels leads to its mineralization and resorption by osteoclasts. The resorption of the cartilagenous matrix signals osteogenic cells, deriveci fiom blood elernents andor hm re- differentiated cartilage cells to replace the cartilage with newly fomed bone. In long bones a proliferative cartilage zone in the growth plate persists and is responsible for the continuhg longitudinal growth of the bones.

D. Molecular Components of the Bone Ma- The collagen fibers in bone are formed fkom type 1 collagen which is the most abundant component of bone, accounting for 70% of the organic material and -90% of the protein. Triple helical molecules of type I collagen aggregate to form fibrils which are arranged into a fïber network which is stable under physiological conditions, and can be seen in polarized light and by electron microscopy (Murray, 1996). While the collagen network cm provide high tensile strength it is unable to resist deformation and alone lacks the ability to maintain bone shape. The incorporation of proteoglycans into the interfbrillar spaces increases the osmotic gradient and provides bone, and cartilage in particular, with the resilience to withstand compressive and shear forces. Further stabilization and the ability to maintain bone shape and form, as well as to create an organized skeleton, is achieved through mineralization. WhiIe type 1 collagen is the most abundant protein in most mammalian connective tissues, the unique qualities of bone matrices cornes tÏom the non-collagenous components. Notably, only bone, dentin and cementurn under nomal physiologie conditions are capable of nucleating hydroxyapatite crystals [Ca,, (PO,), (OH),]. Different mechanisms have been proposed to explain the normal mineraiization process in bone. Although nucleation sites may be provided by the collagen, it seems more likely that non-collagenous matrix molecules like the sialoproteins and proteoglycans control the mineralization process. These highly anionic proteins seem to associate strongly with the mineral phase of the bone (Domenicucci et ai., 1988; Goldberg et al., 1988) and have the ability to act as regulators of crystal nucleation and growth (Kunter et ai., 1996; Boskey, 1995). However, the exact events leading to the mineralization of bone are still obscure. Nevertheless, biological mineralization is recognked to be a tightly regulated process involving both the cellular activity of bone lineage cells and the organic matrix produced by thae ceiis.

II Bone Lineage Ce1 Cultures

A. Models in the Study of Bone Development Bone growth and remodelling are continuhg processes throughout life. Knowledge of the normal sequence of bone formation and remodelhg is necessary for understanding pathologies resulting in a variety of bone diseases, responses to trauma, mechanical loading and the possible phamacokinetic action of or drugs on bone metabolism. Since the formation and remodelling of bone is dependent upon the differentiation of osteogenic cells, a detailed knowledge of bone Lineage cells and their regdation is fundamental to understanding these processes. To shidy osteoblast differentiation and the formation of bone tissues, a number of model systems have been developed as descnbed below. 1. In Vivo Models In vivo observations of avian and rodent skeletal development (Pechak et al., 1986; Smith and Hall, 1990) have hproved our understanding of bone organization and have provided a basis for the initial classification of bone lineage cells according to their location in the bone. For example, the sequence of events in bone fracture healing has been studied using these models (Joyce et al., 1990). However, in vivo models are generally limited by their inability to address signaling events or provide detailed molecular analyses of ce11 differentiation associated with the changes observed in the organ. Thus, while transcription and translation of gene products can be identified with antibodies and by in situ hybridization, it is difficult to study the regdatory pathways that mediate bone formation using in vivo models. However, the intact organ is required for the study of processes that involve an intact vascular system and ultimately observations made in simpler systems must be confïrmed in an in vivo model, especially where clinical implications are concemed. 2. In Vitro Models Bone and cartilage development requires the recruitrnent and differentiation of primitive mesenchpal stem cells (Friedenstein, 1976). The isolation and maintenance of bone tissues and cells in vitro has facilitatecl investigation of early events and restriction points in bone ceU differentiation and is more amenable to understanding modulation mechanisms associated with signaiing and differentiation-associateci events. a) Tissue cultures Tissue cultures have provided the first insights into the conditions needed for the maintenance of bone or cartilage phenotypes in vitro while keeping the ceils in their natural three-dimensional structure. These systems can be used to detect transition stages in bone development when a variety of other ce11 lineages are present in the tissue culture, and cm be used to examine and compare physiological processes in bones fiom different anatomical locations. An example of a useful tissue culture is the chick penosteal system which can be used for studies of osteoblastic differentiation during formation and mineralization of osteoid secreted by periosteal cells in 3- dimensional tissue (Tenenbaum and Heersche, 1986; Tenenbaum et al., 1986). The major disadvantage of tissue cultures is that the study time is restricted by the Limited viability of the cells due to the restricted diffusion of nutrients and metabolites (Luria et al., 1987; Silbennann et al., 1983). b) Primary ce11 cultures Bone marrow cells Bushed from long bones of mamrnals (Owen, 1985; Owen and Fnedenstein, 1988; Till and McCulloch, 1961) offer the possibility of studying bone ce11 differentiation and that occurs in bone tissues. The mmow stroma forms a network of fibroblasts, adipocytes, endothelial cells and macrophages that supports hematopoiesis but also contains cells of the osteoblast lineage (Friedenstein, 1976; Long et al., 1990; Malaval et al., 1994). The osteoblast lineage cells in marrow can be separated f?om the hematopoietic cells by their capacity to attach to culture dishes and grow when supplemented with medium that favours growth of fibroblastic cells. However, the osteoblast Lineage cells appear to represent a maIl proportion of the stroma1 cell populations in human and rodent marrow (Friedenstein et al., 1968; Long et al., 1990). Another commonly used primary culture mode1 is the periosteal-derived ce11 system which is based on the assumption that osteoprogenitors reside in the investing Iayer of the bone (Owen, 1985). Periosted-derived culhues, lachg the hematopoietic ce11 populations, are considered less heterogeneous than marrow-derived cultures. One of the most commonly used models involves release of periosteal ceUs by digestion of 21-d fetal mouse (Yee et al., 1986) or rat calvaria (Bellows et al., 1986; Cohn and Wong, 1979). Periosteal ce11 culture systems have also being developed using human (Robey and Termine, 1985), bovine (Whitson et al., 1984) and avian bones (Berry and Shuttleworth, 1989). Primary cultures of periosteal cells exhibit pluripotentiality indicative of the presence of progenitors in these cultures that can give nse to osteoblasts, chondroblasts, adipocytes and fibroblasts as observed in primary mmw cultures (Aubin et al., 1992). When supplemented with ascorbic acid, these primary cultures undergo a developmentd sequence which is characterized by initial proliferation followed by matrix production and the formation of organized, multilayered tissue nodules that rnineralize in the presence of an organic phosphate (Bellows et al., 1986). The mineralized nodules have a bone-like stnicture that is simila. to the organization of woven bone in fetal calvariae (Lian and Stein, 1992). Moreover, the mineralization pattern of the cultured bone nodules exhibits the same deposition of hydroxyapatite crystals within and between the bundles of type 1 collagen fibrils (Bhargava et al., 1988). c) CeIl lines and transformed cells Clonal ce11 lines (Le. derived nom a single cd) cm also serve as useful tools for studies of osteoblast differentiation (Grigoriadis et al., 1988). There are several cell lines that can fonn a bone-like extracellular matrix, capable of mineralization. For example, the MC-3T3-El ce11 line derived fkom mouse calvaria (Sudo et al., 1983) responds to cytokine stimuli and has comtitutively high alkaline phosphatase expression. In addition, osteosarcoma and other trançformed ce11 lines also exhibit cytokine responsiveness, alkaline phosphatase activity and respond to mechanical loading. Some can also develop a mineralized matrix in vitro, such as the rat osteosarcoma cells Saos-2, UMR-104, UMR-106 (Majeska et al., 1980; Partridge et al., 1983; Rodan et al., 1987). However, transformeci cell lines do not exhibit the hl1 repertoire of differentiation stages seen in primary cultures. Also, characteristicaliy these ceUs exhibit unregulated growth which destroys the normal relationship between proliferation and differentiation and, as a resuit, do not exhibit the 3 defhed periods of bone development in culture as seen in primary bone-ceil culture models (Owen et al., 1990).

B. Developmental Stages in Primary Bone Cell Cultures In osteogenic ce11 culture models, discrete stages in the development and formation of osteoblastic cells have been identified (Lian and Stein, 1992; Owen et al., 1990) that correspond to developmental changes during bone formation in vivo (Aronow et al., 1990). nie growth and differentiation of bone lineage cells leads to the production of bone matrix, which eventually rnineralizes. The temporal expression of specific genes in the fetal rat calvarial ce11 cultures (FRCC) has been shown to refiect the three main phases of proliferation, matrix production and rnineralization (Owen et al., 1990), as described previously. During the prolifeation period plated cells are sparse with no cell-to-ce11 contacts, and cells undergo repeated divisions. The expression of early bone lineage markers cm be detected at the end of this period (Aubin et al., 1992). As the culture matures, secretion of mate proteins becorne predominant, during the ma& formation stage which occurs fkom day 4 to day 10 of the cultures. There is a dom-regulation of DNA synthesis and an increase in the expression of type I collagen and TGF-P which are related to matrix production. These marken also represent early events in bone lineage ce11 differentiation. The extracellular matrix composition undergoes stages of maturation with the sequentiai deposition of bone proteins. The increase in the bone matrix proteins by the end of the matrix synthesis period is temporally related to the multilayering of the cells with the formation of nodular clusters that incorporate calcium and phosphate ions to form organized masses of hydroxyapatite crystals. Active rnineralization that occurs during this stage is indicative of differentiated osteoblasts in the cultures (Lian and Stein, 1992; Yao et al., 1994). The temporal expression of bone matrix proteins observed in vitro (Ibaraki et al., 1993; Lian and Stein, 1992; Owen et al., 1990; Yao et al., 1994) are consistent with observations in vivo (Chen et al., 1993; Chen et al., 1992; Yoon et al., 1987). Consequently, matrix proteins can, potentially, be used as markers of osteoblast development during the maturation of osteogenic cultures.

C. Bone Matni Proteins and Culture Maturation Two major processes in the maturation of osteoblast heage cells are proliferation and differentiation. In primary cultures of FRCCs a reciprocal relationship between these processes has been suggested (Lian and Stein, 1992; Owen et al., 1990) fiom the temporal sequence of the transcription of proliferation-associated and matrix- associated genes. Thus, the synthesis and accumulation of bone proteins by the osteogenic cells increases only at the end of the proliferative stage. These non- collagenous bone matrix proteins, which serve to characterize this lineage, are expressed during the matrix synthesis stage when differentiation events are most readily observed. Proteins that are expressed in early proliferation stage Uiclude the growth-associated protooncogenes c- fos and c-myc and the H4 histone genes, which reflect the increase in DNA synthesis (van Wijnen et al., 1991). Down-regulation of proliferation is associated temporally with the increased transcription of akaline phosphatase, type 1 collagen, fibronectin and TGF-P, proteins that are al1 associated with matrix production. The expression of type 1 collagen in particular exhibits a reciprocal relationship with the proliferation process. If included in the matrix at early stages of these cultures it will inhibit proliferation and induce early differentiation (Lynch et al., 1995). Conversely, fi'bition of collagen synthesis by ascorbic acid deprivation will prevent continuous growth and Merentiation of cells in culture (Owen et al., 1990). Alkaline phosphatase and osteopontin are two of the earliest markers expressed in osteoblast differentiation and are easily detected at the end of the proliferation stage and the begirining of the matrix synthesis stage (Aubin et al., 1992; Lian and Stein, 1992; Yao et al., 1994). However during rnatrix formation, alkaline phosphatase activity increases during the differentiation process (Lian and Stein, 1992) suggesting that alkaline phosphatase activity may be related to alterations in the rnatrix in preparation for the mineralization process. However expression of osteopontin declines during this time pex-iod (Lian and Stein, 1992; Yao et al., 1994) but is expressed again at higher levels during the mineralkation phase. The ktosteoblast marker expressed at the beginning of the rnineralization phase is bone sialoprotein (BSP), and is incorporated into the mineralized matrix (Chen et al., 1992; Yao et al., 1994). In contrast to OPN, which is bound to the minerd crystals, BSP is also associated with the collagenous rnatrix, as well as with the newly-forrning mineral crystals (Kasugai et al., 1992). Based on the temporo-spatial synthesis of BSP in bone fornation in-vitro (Kasugai et al., 1992; Yao et al., 1994) and in-vivo (Chen et al., 1994; Chen et al., 1992), and its ability to promote the formation of hydroxyapatite crystals in-vitro (Hunter et al., 1996), BSP is thought to initiate the mineralization of newly-formed bone (Sodek et al., 1992). Although, osteopontin has similar physico- chernical propertïes as BSP, its quantitative binding to pre-formed mineral crystals in bone (Kasugai et al., 1992) and its ability to inhibit hydroxyapatite crystal formation and growth (Hunter et al., 1996) indicate that it regulates rnineralization (Sodek et al., 1992). Another marker of mature osteoblasts is osteocalcin which can also regulate mineralization and couple bone formation and resorption in the remodelling process (Aronow et al., 1990; Hauschka et al., 1989). As mineralization proceeds, alkaline phosphatase and type 1collagen transcription are reduced (Lian and Stein, 1992). Notably, the temporal expression of bone proteins that has been observed in fetal rat calvariai and bone marrow cultures reflects the expression patterns of the same proteins in vivo (Chen et al., 1993; Chen et al., 1992; Weinreb et al., 1990; Yoon et al., 1987). Consequently, these cultures systems are considered appropriate for studies of osteoprogenitor differentiation and bone formation.

III Bone Lineage CeUs

A. Definitions and stages Cells derived fiom bone tissues are a heterogeneous population, including osteoblasts, chondroblasts, fibroblasts, myoblasts, and adipocytes, that are believed to originate f?om a common progenitor ce11 (Aubin et al., 1995; Owen, 1985; Owen and Fnedenstein, 1988). However, the series of differentiation events that lead to the formation of the specialized ce11 populations are still largely obscure. The mature osteoblast is a cuboidal ce11 characterized by a well-developed endoplasmic reticulum, large spherical Golgi complexes and extensive cytoplasmic processes which in vivo, extend into the osteoid matrix (Cormack, 1987; Murray, 1996). Active osteoblasts have high aikaline phosphatase activity, abundant receptos for parathyroid hormone (Aubin et al., 1988) and exhibit rapid synthesis of type 1 collagen, osteocalcin, OPN and BSP. At the end of the secretory penod, the volume and the number of organelles associated with synthetic activity are reduced. When osteoblasts remain on the bone surface they are described as lining cells. The osteocytes, which provide cellular continuity througout the mineralized matrix, are eventually digested during the remodelling process (Murray, 1996). Cells in the pre- osteoblastic stage are relatively small with poorly-developed cytoplasmic organelles. They can be identified in bone tissue sections fiom their location adjacent to active mature osteoblasts and lining cells (Comack, 1987; Holtrop, 1975), while in culture they exist as a preosteoblastic population that is prevalent in early stages (Le. proliferation stage) of culture (Lian and Stein, 1992; Owen, 1985). B. Early Precursor Cells of Bone Stroma1 cells derived from bone include cells with a high proliferative capacity and rnultipotentiality, characteristics that are indicative of the presence of stem cells (Owen and Friedenstein, 1988). Osteogenic stem cells have not been identified yet due to a lack of phenotypic markers, low abundance or any known specific synthetic activity (Aubin et al., 1995). Alkaline phosphatase positive populations that exhibit high proli ferative capacity, but lack self-renewal ability are classified as osteoprogenitors (Turksen and Aubin, 199 1) whereas in other lineages comparable ce11 populations are refened to as transit amplifying cells (Potten and Loeffler, 1990). In bone marrow, the majority of cells are hematopoietic iineage cells, while the preosteoblastic population is thought to originate fiom a mixed fibroblastic population of cells (Murray, 1996). Preosteoblastic cells may also reside in the investing layer of the bone (Owen, 1985). Irradiated animals treated with mmw transplants can regenerate their hematopoietic marrow environment ushg donor cells while the bone- forming cells are derived fiorn the endosteal surfaces of the recipient (Friedenstein et al., 1978). This provides further evidence for the existence of bone stem cells in the investing layers. The presence of stem cells in bone ce11 cultures can be demonstrated by the ability of a single ce11 to profiferate and to differentiate into osteoblasts, choiidroblasts, myoblasts and fibroblasts (Gngonadis et al., 1 988). However, due to our lack of complete understanding of osteoblast functions and the lack of markers for early precursor cells in these lineages, Little is known about early progenitors. The use of osteogenic markers such as alkaline phosphatase and non-collagenous bone proteins identifies cells already committed to the osteoblast pathway but not the progenitor cells. In primary bone ce11 cultures the tirne when cultures are enriched for osteoprogenitors has been dehed (Aubin et ai., 1992; McCulloch and Tenenbaurn, 1986). At early times of culture development (24-72h), cells exhibit a high proliferative capacity and expression of osteoblast differentiation markers is low. However, due to the heterogeneous populations in primary cultures, it has not been possible to isolate the osteogenic progenitor celis for detailed characterizaîion. The identification and nomenclature used for the description of bone heage cells has been derived from an early dennition by Friedenstein (Friedenstein et al., 1974). The inducible osteoprogenitor ceils (IOPC) are self-renewing and at each ce11 division another IOPC is formed. Given the appropriate stimuli, IOPC's have the capacity to form a determined osteoprogenitor @OPC) that can differentiate into a mature osteoblast. Many hypothetical models describe the series of events leading nom the pluripotential bone progenitor cells to more differentiated cells that will eventually lead to the bone forming ceil (Aubin et al., 1995; Cormack, 1987; Lian and Stein, 1992; Owen, 1985), however the precise series of molecular signals and phenotypic changes involved have yet to be determined.

C. MethocLFfor Characterizkg Early Precursors in Bone Lineage Cell Populations The study of bone cells has been hampered by the lack of unique phenotypic markers for the different subpopulations in this lineage. For osteoprogenitors the problem is Mercomplicated by their low fkquency (0.006% in marrow cultures (Falla et al., 1993); 0.3% in periosteal cultures (Bellows and Aubin, 1989). Moreover morphological criteria alone can not be used to identiQ osteoblastic subpopulations. Thus, in a recent study, phenotypic heterogeneity has been documenteci in cells with the same morphological characteristics (Liu et al., 1994). Cells with différent phenotypes have been separated using density centrifugation (Villanueva et al., 1989) but no information concemuig the recovery of osteoprogenitors is available. CeIl cloning procedures are another valuable approach which demonstrate indirectly the presence of progenitor cells that cm differentiate into more than one ceil type (Grigoriadis et ai., 1988; Partndge et al., 1983). This approach is especially useful in studying the effect of various cytokines on bone cell differentiation. However it has not yet been determined whether clonal cells undergo al1 the differentiation stages that occur in nomal bone culture development and it is unclear if they are tmly representative of naturd osteoprogenitors. The use of monocIonal antibodies agauist bone specific proteins to detect and isolate different subpopulations in the odeoblast lineage has been successfid for the study of the cornmitted osteoprogenitor cells (Haynesworth et al., 1992; Turksen and Aubin, 1991). This approach codd be a valuable method for isolating osteoprogenitor populations when unique markers can be identified. It has been suggested that stem cells in general are not a homogenous population but instead are a mixture of progenitor subclasses, which need mercharacterization before preparing stage- specific antibodies (Potten and Loeffier, 1990). Flow cytometry analyses and sorting of immunocytochemicaiIy stained cells has been used to detect expression of bone lineage markers but the poor viability and Iow numbers of recovered cells has inhibited Mer development (Turksen and Aubin, 1991; Van Vlasselaer et al., 1994). The use of flow cytometry has been more successfûl when the isolated cells exhibited bone differentiation markers; these markers helped in Mercharacterization of the precornmited cells in this lineage (Horowitz et al., 1994; Long et al., 1995). Notably, most of the methods used for bone lineage studies have been derived fiom methods used in the shidy of the hematopoietic Iineage. However, hematopoietic cells do not exhibit dependence on matrix synthesis, whereas bone ce11 di fferentiation is a matrix-dependent process. Conceivably, several of these rnatrix proteins could be used as osteogenic ce11 lineage markers. One of these proteins, osteopontin (OPN), appears to be highly expressed at two stages of osteogenesis: at the early, proliferative stage and at a later stage, subsequent to the initial formation of mineralized bone matrïx (Lian and Stein, 1992; Yao et al., 1994). The early expression of OPN is consistent with its immunolocalization in pre-osteoblastic cells, indicating that this protein could be an early indicator of osteogenic differentiation (Mark et al., 1987; Mark et al., 1987; McKee and Nanci, 1996). IV Osteopontin

A, Stnrcr~re Osteopontin (OPN) was first identined in transformed ce11 lines as a secreted 60- kDa protein by Senger et al. (Senger et al., 1979) and termed "transformation-specific phosphoprotein". It was later re-discovered as a transformation-associated ceH-adhesion protein by molecular cloning and named 2ar (Craig et al., 1989). The same gene product was identified as a putative lymphokine produced by activated and macrophages and caiied eta- 1 (Cantor, 1995). However, the protein was independently identified as a sidoprotein in the extracellular ma& of bovine bone by Fmén and Heinegard (1985), who initiaily caiIed it bone sialoprotein 1 (BSP I) to distinguish it fiom a second sialoprotein, BSP II, now known as bone sialoprotein (BSP). More detailed characterization of the rat protein, described as a 44kDa phosphoprotein, was reported by Prince et al (1987). Following elucidation of the prirnary structure of rat OPN, determined fiom a cDNA cloned hman osteosarcorna ce11 line (ROS 17/2.8) library (Oldberg et al., 1986), the name osteopontin was coined to refiect the potential of the protein to bndge between bone cells and hydroxyapatite. Although a new name, secreted phosphoprotein 1 (SPP I), was introduced to recognize the broader îûnctions of the protein, the name osteopontin has been retained following the nomenclature used for the identification of the human gene (Denhardt et al., 1995). 1. Characten'stics of the Protein The primary sequences of OPNs nom rat (Oldberg et al., 1986), mouse (Craig et al., 1989), pig (Wrana et al., 1989)- human (Kiefer et al., 1989), cow (Kerr et al., 199 1) and chicken (Moore et al., 1991) have been detennined fiom corresponding cDNAs. The mRNA transcript of the rat OPN is -1.4 kb and includes the leader sequence as well as the AUG initiation codon. Analysis of the nucleotide sequence revealed that the protein is rich in aspartic and glutarnic acids and serine and contains a polyaspartic acid motif, through which the protein can bind to hydroxyapatite, and an RGD sequence which can mediate ce11 attachment (Oldberg et al., 1986). In addition, multiple sites of serine and threonine phosphoryIation and sites of both N- and O-linked glycosylation were ident5ec.i together with a potential hmbin cleavage site. Cornparison of mammalian OPN sequences reveaied 50% sequence identity and conservative replacements in an additional 10% of the amino acids (Crosby et al., 1996; Sodek et al., 1995). In cornparison, the avian sequence shows only a few conserved regions when compared with the mammalian sequences. However, these include the polyaspartic acid sequence, the RGD ce11 attachment site, multiple serine phosphorylation sites, and the thrombin cleavage site (Sodek et al., 1995). The characterization of the rat bone OPN has show that signincant post- translational modification of the nascent protein does occur (Rince et al., 1987). Twelve phosphoserines and a phosphothreonine together with 5-6 O-Iuiked and a single N- linked oligosaccharide are present in the bone protein. However, significant structural variation which can occur through post-translational modifications is evident in OPNs fiom the same and different tissues (Denhardt and Guo, 1993; Sodek et al., 1995). Moreover, while the mammalian and avian proteins have a similar number (-317) of amino acids, the size of the secreted protein can Vary fiom 44kDa to 75kDa on SDS- PAGE gels. AIthough these differences can, in part, be attributed to anomalous behaviour on SDS-PAGE, variations in phosphorylation, glycosylation and sulphation have been reported (Kubota et al., 1989; Nemir et al., 1989; Sin& et al., 1990). In addition, multiple forms of OPN have been identified as products of normal (Kasugai et al., 1991 ; Kubota et al., 1989) and transformed cells derived nom rodent tissues (Craig et al., 1989). Rat bone cells produce high and low-phosphorylated forms of OPN, the high phosphorylated Corn being associated with differentiated osteoblasts (Kubota et al ., 1989; Sodek et al., 1995). In transformeci cells, two forms of phosphorylated OPN, termed pp62 and pp69, and a non-phosphorylated form np69 have been characterized (Nemir et al., 1989). Although the physioiogical significance of these foms is not clear, it is Iikely that the differences will reflect different roles, as indicated by diBerences in their functional properties. Thus, while pp69 has been shown to associate with ce11 surface fibronectin, which requires the presence of sialic acid on the oligosaccharide side chains (Skuunugam et ai., 1997), the non-phosphorylated np69 immunoprecipitata with soluble fibronectùl (Singh et al., 1990), whereas the pp62 remains unbound. Also, a clone of mouse epidermal ceils that secretes non-phosphorylated OPN upon induction with calcitriol, secretes a phosphorylated fom of OPN foilowing malignant transfo~nationinduced by the tumor promotor 12-O-tetradecanoyl phorbol-13-acetate ( TPA) (Chang and Rince, 1993). Phosphorylation of OPN can take place on tyrosine residues, or on serine and threonine residues involving catalysis by several protein kinases (Saavedra et al., 1995; Salh et al., 1995; Salih et ai., 1996). These include a factor-independent protein kinase pK)(Ashkar et al., 1993) such as casein kinase II (Patarca et al., 1993), go@ kinases, cAMPdependent kinase, and protein kinase C. Celi surface phosphorylation of OPN has also been reported (Zhu et al., 1997) while recombinant nascent OPN can cataiyze autophosphorylation of tyrosines (Ashkar et al., 1993; Saavedra et al., 1995). Notably, compared to rat bone OPN which has twelve phosphoserines and one phosphothreonine (Prince et al., 1987), bovine rnik OPN contains 27 phosphoserines and one phosphothreonine with a different pattern of phosphorylation than the rat OPN (Sorewen et al., 1995). Thus, there is considerable potential for phosphorylation reactions to modi@ the structure and properties of OPN and to have a possible involvernent in signalling mechanim. Significant glycosylation of OPN is indicated hmthe size of the molecule, estimated at >33% more than the weight of the protein backbone (Oldberg et al., 1986). Furthmore, treatment of OPN with glycosidases reduces the size of the protein markedly on SDS-PAGE (Shanmugarn et al., 1997). In addition to the single Am-X-Ser site for N-linked glycosylaiion there are 26 Ser-X-Glu sites for O-Linked glycosylation. in transformed cells the non-pnosphorylated fom of OPN contains N-linked carbohydrates (Singh et al., 1990) which may prevent phosphorylation reactions. OPN sulfaton has been observed in the mineraikation process of bone-nodules by fetal rat calvarial cells. Sulfation of OPN occurs predorninantly in the highly- phosphorylated form of OPN and this has been suggested as a potential marker for differentiated osteobiasts (Nagata et ai., 1989). OPN sulfation was shown to be in the fom of tyrosine date residues (Ecarot-Charrier et ai., 1989), however potentiai

dation sites can also OCCUT in the oligosaccharide side chains (Singh et ai., 1990; Bang et al., 1990)

B. Regdation of OPN Erpression 1. Hormonal and Cytokine Control of OPN Synthesis. Both 1,25dihydroxyvitamin D3 and giucocorticosteroids Uicrease OPN expression in bone celis. Whereas glucocorticoids inhibit OPN expression in fibroblastic cells (Nomura et al., 1989) the synthetic glucocorticostemid, dexamethasone, has been shown to increase OPN expression in rat bone cell cultures (Nagata et al., 1989; Oldberg et al., 1989; Yao et al., 1994) and in cultures of embryonic rat bones (Chen et ai., 1996). Vitamin D3 is a particularly potent shulator of OPN synthesis (Noda et al., 1990). However, the increase in OPN induced by glucocorticoids is Wed to the stimulation of bone formation whereas the vitamin D3 ektis associated with massive bone resorption (Chen et al., 1996), reflecting the multifùnctionaiity of OPN in bone remodelling (Sodek et al., 1995). Of the other osteotropic homones, retinoic acid has been shown to increase OPN expression whereas PTH has a marginal effect that appears to be bone ce11 type- dependent (Kasugai et al., 1991). OPN transcription is upregulated by epidermal growth factor (EGF) in NRK cells, bone cells and endothelid cells (Kasugai et al., 1991; Senger et ai., 1996). Other growth factors such as PDGF, TGF-B, together with non-physiological agents such as TPA and concanavalin A, also increase OPN expression; the magnitude and nature of these effects are cell-type dependent (Kasugai et al., 1991). Bone morphogenetic proteins, such as BMP-7, which stimulate bone formation by promothg osteoblastic differentiation, directly increase OPN transcription in FRCC cultures (Li et al., 1996). 2. The OPN Gene and Transcriptional Regulation. Osteopontin is encoded by single copy gene which has been identified in human, mouse and rat. The mouse gene is locaiized to chromosome 5 at the locus of the rickettsia resistance gene, rie (Fet et al., 1989; Miyazaki et al., 1989), whereas the human gene is localized to the long arm of chromosome 4 (4q13) (Hijiya et al., 1994) close to the BSP and DMP- 1 (Dentin Matrix Protein-1 ) genes (Aplin et al., 1995). The mouse gene comprises seven exons and five introns that span approximatety 4.8 kilobases. Exon 1 contains the 16 amino acids of the leader sequaice. Exons 2, 3, 4, 5, and 6 encode 12, 27, 14, 94, and 129 amino acid residues, respectively. Exon 5 encodes regions containing 10 consecutive Asp mino acid residues and the Gly-kg-Gly-Asp Sapeptide. Exon 6 encodes the C-terminal halfof OPN. The human gene contains 7- exons which are sirnilar to those of the mouse gene but span -1 1.1 kb. The ciifference in length is mainly due to variations in intron 3, which is approximately 2.7-fold longer than that of the mouse OPN gene (Hijiya et al., 1994). Approximately 1 kb of the rnouse promoter region has been sequenced and analyzed for potential transcription factor recognition sites (Craig and Denhardt, 1991). A TATA box is present at position -28 to - 22, an inverted CCAAT box at position -55 to -50 and a GC box at position -100 to -93. A fiuictional 125-dihydroxyvitamin D3 response element (VDRE) is present f.urther upstream (nts -758 to -741) of the immediate promoter region (Noda et al., 1990). In addition, activator protein (AP) motifs were also identifieci when the regulation of OPN transcription was studied with the tumor-promoting phorbol ester, TPA (Denhardt and Guo, 1993). The sites identified include: AP-1, AP-2, AP-4, and AP-5. AP-1 sites are highly conserved elements controiled by the proto-oncogene products Fos and Jun. In transformed rodent cells the activity of AP-l/PEA-1 is stirnulated by a serum component, TM, v-src and retinoic acid. AP-2 mediates signal transduction involving protein kinase C and CAMP-dependentprotein kinases A. There are 5 recognition sites for the polyoma enhancer activator PEA-3. These sites are the major targets of the Ets family of proto-oncogene transcription factors that are involved in T-ce11 activation and could, therefore, account for the up-regulation of OPN expression in activated lymphocytes and macrophages (Denhardt and Guo, 1993). Following the observation that OPN expression is dom-regulated in src 4-ve transgenic mice (Chackalaparampil et al., 1996), which are characterized by an osteopetmtic phenotype (Sonano et al., 1991), regdation of OPN by src was subseqyentiy shown to increase OPN expression through the invertecl CCAAT box (Tezuka et al., 1996). Since v-src is a transforming Wal oncogene product, originaüy identified in the Rous sarcoma virus (RSV), these observations provide a £irm link betweem cellular transformation and induction of OPN expression. Similarly, cells transfomed with v-ras and v-rnyc dso increase transcription of OPN (Chambers, 1992; Chambers et al., 1992), dthough the mechanis- of induction has not been determineci for these oncogenes. While the significance of OPN expression in tumor ceUs has not been established, the increased transcription of OPN observeci in lung tumors correlates with tumor progression and could be used to predict the tumor prognosis (Chambers et ai., 1996). 3. Altemate Spliced fomof OPN mRNA Two forms of OPN mRNA, that differ by the presence or absence of 14 amino acids beginning at residue 58 of the molecule, have been identified in human bone and decidual cells (Young et al., 1990) and three types of splice variants have been observed in human glioma tumors (Saitoh et al., 1995). Analysis of cDNAs prepared fkom Kirsten sarcoma virus-transfonned NRK cells (KNRK) and ROS 17/2-8 rat osteosarcorna cells have revealed a 52-nucleotide insert in the 5'- non-coding region of the KNRK OPN cDNA (Si@ et al., 1992). While the alternately spliced forms expressed by the human cells can give rise to different proteins the altemate splicing in the rat transcripts would not alter the translated protein.

C. Tissue Distribution and Proposed Biological RoZes OPN is recognized as a prominent bone matrix protein that is produced by osteoblastic cells. Although not exclusive to bone, OPN has been suggested as a usehl marker for osteoblast differentiation (Aubin et al., 1992). The early expression of OPN during bone formation is consistent with its immunolocalization in pre-osteoblastic cells, suggesting that it could be an early indicator of osteogenic differentiation (Mark et al., 1987). Following secretion, OPN is found in the osteoid and in the mineralized bone ma& and is also present in mineraiized cartilage (Chen et al., 1991; McKee and Nanci, 1996). In remodeiiing cortical bone, seams of enriched OPN staining have been observed at sites of cernent formation and in lamina limitans structures beneath lining cells, which may represent early and late events in bone formaton (Chen et al., 1994; McKee and Nanci, 1996). This is supporteci by the biphasic expression of OPN during osteogenesis observed in vivo (Chen et al., 1993; Yoon et al., 1987) and in vitro (Lian and Stein, 1992; Yao et al., 1994). Notably, the early expression of OPN in bone marrow cultures (Yao et al., 1994) has been correlated with the formation of a cernent layer (McKee and Nanci, 1996). The later expression of OPN appears to be important in the regdation of hydroxyapatite crystal growth and the formation of the limitans layers beneath linùig cells and surrounding osteocytes where it can mediate the attachent of these cells and during bone remodeling, mediate the attachent of osteoclasts (Dodds et al., 1995; Reinholt et al., 1990). The OPN gene is also expresseci by cells in non-minerahhg tissues including kidney and specialized epithelia, and by activated lymphocytes and macrophages and by transformed cells (Denhardt and Guo, 1993). Although the precise function of the OPN in any of these tissues is unknown it is evident that OPN can influence cellular and humoral immune responses (Cantor, 1995). The unique conserved regions of the OPN molecule (i.e. RGD site, a thrombin cleavage site, polyaspartic acid sequence and serine phosphorylation sites) may explain some of these extraskeletal properties. The polyaspartic acid sequence and serine phosphorylation sites that appear to be involved in regdating rnineralization of bone rnay act in a similar manner in other calcified sites. Thus the presence of OPN in calcified blood vessels could be related to a role in mineralization [Shen, 1997 #33 1; Mohler, 1997 #292] but the mechanism that triggers OPN expression in the endothefial cells is &om. The RGD sequence can mediate ce11 attachrnent and ce11 migration, as well as intracellular signaling through the a$, and other integrins (Liaw et al., 1995; Miyauchi et al., 1991).The signahg by OPN through a$, leads to cytoskeletal rearrangement associated with either tyrosine phosphorylation (-ka et al., 1995) of actin binding proteins or intracellular calcium changes (Zimolo et al., 1994). The importance of integrins in the attachrnent of bonedenved fïbroblastic cells was oripinaily reported in studia by Somman et al. (1987) in which concentrations as low as lpg/ml of OPN were found to be as effective as fibronectin in promoting ceU attachment and spreading. Moreover, the attachment could be blocked by RGD-containing peptides (Somerman et al., 1989). Subsequently, OPN was shown to be highly enriched at osteoclast attachment sites in bone resorption bays and shown to codisûibute with vitronectio receptors on osteoclast membranes (Reinholt et al., 1990) and osteoclast attaEhment and resorption capacity were shown to be inhibited by antibodies to qP3 in chicken osteoclasts (Miyauchi et al., 1991). In more recent studies, dephosphorylation of OPN has been iinked with loss of attachment properties supporting a mechanism for osteoclast amchment (Horton et al., 1995). However in other studies the validity of the hdings showing osteoclast attachment through OPN-a$, has been questioned (Flores et al., 19%). OPN involvernent with ce11 motility and adhesion in attachment and spreading was also seen in regenerating endothelium and smooth muscle cells (Liaw et al., 1995) where recombinant osteopontin stirnulated adhesion and directed migration of bovine aortic endothelid cells. In rat smooth muscle cells OPN enhanced migration in a time and concentration-dependent marner (Yue et al., 1994), as dso seen in placental trophoblasts (Daiter et al., 1996) and kidney cells (Rabb et al., 1996). OPN effects on ce11 spreading and attachment were shown to be inhibited by soluble RGD peptides (Senger et al., 1994). Notably, thrombin cleavage of OPN amplifies the attachrnent and spreading properties induced by OPN. The presence of a thrombin cleavage site which is 6 amino acids from the RGD site seems to ailow greater accessibility to the RGD site and cleavage of OPN increase the inductive effects mediated through the a$, integrin receptor (Senger and Pemipi, 1996). The thrombin cleavage site and the ability of OPN to serve as a substrate for the coagulation factor plasma transglutaminase (Factor Xma) are suggestive of a role for OPN in wound healing (Rince et al., 1991). OPN cm also down-regulate transcription of nitric oxide synthase which in activated macrophages gaierates niûic oxide. This effect can be blocked with RGD containhg peptides indicating that OPN si@ through an integrin receptor (Rollo et al., 1996). Moreover OPN was shown to reduce the cytotoxicity of secreted nitric oxide. Thus the increased expression of OPN in transformed and metastatic cells may protect these cells against cyto toxicity (Rollo and Denhardt, 1996). Aithough the RGD site and its association with the a,,integrins could explain some of the attachment properties of OPN, rnediation of ce11 attachment and adhesion by OPN without involving integrin receptors has also been reported (Weber et al., 1996). Thus, a rh-OPN lachg the RGD site cm efficiently promote ce11 attachent of fibroblastic and melanoma cell lines (Katapiri et al., 1996). Moreover, RGD peptides did not alter OPN attachment to these ceils, suggesting that some cells can interact with non-RGD binding sites in OPN. Thus, OPN has been shown to be a ligand for CD44 which is characteristically expressed in rnigrating immune and tumor cells and is the main receptor for hyaluronan (Weber et al., 1996). In con- to hyaluronan, which prornotes ce11 aggregation through its binding to CD44 OPN stimulates chernotaxis and ceil migration. Since expression of CD44 (Naot et al., 1997) and OPN (Chen et al., 1997; Senger et al., 1979) have both been Linked to turnor formation and metastasis, this interaction may shed light on the role of these proteins in normal and pathogenic ce11

D. OPN Signals nirough Integrim and CD44 1. Integrins Integrins are non-covalently associated heterodimers fomed fiom two (aand P) protein chains (Hynes, 1992). Most cells express several integrin receptors which recognize different attachment molecules through RGD and other conserved motifs. The interaction of OPN with integrins has been well investigated in several ce11 types. OPN and OPN-denved RGD-containing peptide interact in a Ca" dependent manner with a$,,, and a$, integrins (Liaw et al., 1995; Smith et al., 1996). The a$, is prirnarily responsible for the adhesion properties of OPN (Ashizawa et al., 1996; Miyauchi et al., 1991) and in some cells is believed to generate intracellular signals based on the structural features of the Ligands (Senger et al., 1994). OPN has dso been shown to modulate iniracellular Ca2' througb Ligation to a$, (Zimolo et al., 1994). The fact that more than one heterodimeric combination can rnediate OPN ligation may account for different signals that OPN can elicit in cells, as observed for other attachment proteins (Hynes, 1992). Thus, integrin receptors appear to play an important role in attaching cells to OPN in the extracellular ma& and also mediate some of the intracellular signaling associated with the attachment of cells to OPN.

2. CD44 CD44 is a transmernbrane glycoprotein known to bind extracellular matrix components such as hyluronan (Hale et al., 1995; Haynes et al., 1991; Patel et al., 1995). It has been extensively studied in the last few years due its proposed role as an immune cell-surface marker (Koshiishi et al., 1994; Naot et al., 1997). CD44 is expressed by a variety of ce11 types including lymphocytes, myeloid cells, fibroblasts, epithelial cells, endothelid cells, muscle cells, and astrocytes in the central nervous system. A strong interest in CD44 its expression in malignant transfomation of cells (Naot et al., 1997). The expression in tumors is variable and changes in relation to hunor invasiveness properties and prognosis (Kaaijk et al., 1995; Nagabhushan et al., 1996; Sy et al., 1996; Tanabe et al., 1995). The CD44 gene produces a large number of isoforms by alternative splicing of at least 10 exons, specifically exons 6 to 15+ which are often cdled vl-vlO. AI1 the modifications relate to the proximal extracellular domain. So far 100 isoforms have been documented, resulting fkom a high nurnber of splice combinations. The most prevalent form of CD44 is a 85-95 kDa isofonn which has a 40kDa peptide backbone with carbohydrate present on numerous glycosylation sites as oligosaccharides or glycosaminoglycans. The extracellular N-terminal region of CD44 contains two binding sites for hyaluronic acid while the C-terminai region comprising the cytoplasmic domain has two putative binding sites for ankynn proteins and four potential sites for serine phosphorylation. CD44 is thought to mediate adhesion and aggregation of ceiis to hyaiuronate (Bartolazzi et al., 1996; Koshiishi et al., 1994) through two or more sites (Bartolani et al., 1996). It has been suggested that clustering of CD44 on celi surface may be important for adhesion through multiple interactions with individual hyaluronan molecules (Underhill, 1992). Hyaluronan binding is not a trait cornmon to ail CD44 isofomis and it has been shown that expression of CD44 isofomis that bind hyaluronate augments the rapidity of turnor formation by melanoma cells in vivo (Bartolazzi et al., 1994). Hyaluronan binding capacity is also linked with the CD44 cytoplasmic domain and it had been shown that the lack of 57 amino acids fkom the C-terminal region inhibits binding to hyaluronan (Liu et al., 1996). CD44 expression is also associated with increased cell motility (Kaaijk et al., 1995; Sy et al., 1996; Zhu and Bourguignon, 1996). For example, induction of apoptosis leads to CD44 shedding and the loss of motility (Gunthert et al., 1996). Also, the injection of anti-CD44 antibodies into animals carrying metastatic tumors cmeliminate metastases (Seiter et al., 1993). The relationship between CD44 induction and iocreased cell rnotiiity and ce11 signalhg could be mediated by its connection to actin-binding proteins like the ezrh radixin moesin (ERM) family of proteins (TsulQta et al., 1994). This family of proteins is known to link ce11 surface adhesion molecules to actin filaments in the subcortical area and thereby serve as membrane cytoskeleton linkers. Moreover, these proteins may facilitate actin filament assembly and transduction of signais through tyrosine kinases or G-proteins (Beriyman et al., 1995; Takaishi et al., 1995). The association of the intracellular domain of CD44 with the ERM family is well established in fetal cells or cells exhibiting rapid migration properties such as hunor cells. Hyaluronate ligation by CD44 may mediate signal transduction through ERM or gelsolin (Chellaiah and Hniska, 1996), actin polymerization and increased ce11 motility. The temporal and spatial expression of CD44 seems to correlate with OPN expression, especially in processes where transformation of cells occurs. Transfection experiments with OPN, even in benign tumors, cm induce metastatic potential (Chen et al., 1997). In a simila. manner transformation occured when benign epithelial tumors transected with CD44v (Iida and Bou~guignon,1997). Moreover, epidemal growth factor EGF (which upregulates several proto-oncogenes such as c-rnyc or Met (Chen et al., 1997; Onodera et al., 1997)) seems to sïmultaneously upregufate ezrin, CD44 and OPN (Berryman et al., 1995). The coexpression of OPN and CD44 in invasive tumors was explained by a possible autocrine fiuiction for OPN in the secretory ceils (Weber et al., 1997). However, in rapidly migrahg cells it seems unIikely that CD44 and secreted extracellular OPN are Linked in the same pathophysiological process. Statement of Probiem

Osteogenic cultures contain cells of different stages of differentiation. This hetemgeneity has preciuded a rigorous examination of the ongin of osteogenic cells and the developmentd stages of osteoblast differentiation. Moreover, single-cell analysis of the phenotypic characteristics of osteogenic cells has indicated that osteogenic preçursor cells may also be heterogeneous (Liu et al., 1994). Nevertheless, atternpts have been made to separate osteoprogenitor and osteogenic cells using density centrifugation (Villanueva et al., 1989) and flow cytometry, using ceii surface markers (Long et ai., 1995). However, successful application of these appmaches has been Limited by the relatively poor viability of the sorted cells. Moreover, positive selection is more likeIy to select for relatively differentiated cells, which have already begun to express phenotypic markers associated with osteoblastic di fferentiation (e.g. aikaline p hosp hatase; (Turksen and Aubin, 1991)). Since OPN is expresseci early in bone formation (Mckee et al., 1995; Sodek et al., 1995) and has been suggested to be a usehl marker for osteoblast differentiation (Aubin et al., 1992), I hypothesized that when used in combination with flow cytometry the expression of osteopontin might be used to analyze osteogenic diflerentiation when applied to cultures of fetal rat calvarial cells. Based on results obtained hm experiments designed to test this hypothesis it was Merhypothesized that the absence of OPN expression in mal1 cells with low cytoplasmic grandarity was indicative of early precurçor cells with the ability to differentiate into osteoblasts and other lineages. The observation that intracellular OPN staining generated two distinct patterns, one of which was characterized by focal aggregates at the ce11 surface provided the basis of the third hypothesis, that this perimernbranous staining pattern was associated with a migratory phenotype in which the OPN was associated with a specialized attachrnent complex. Based on sirnilarities in the expression of OPN and CD44 in nomal and transformed cells and the demonstrated association of OPN with this receptor, the CD44 cell attachent system was considerd as a possible site for extracellular OPN. Based on these hypotheses the objectives were as follows: 1) To use intraceildar OPN immunostaining and flow cytomeûy to analyse stages in osteogenic Merentiation in cultures of fetal rat calvarial cek. 2) To isolate a population of mail OPN-ve cells, with low granularity, and characterize these cells with respect to their anticipateci potential as early osteogenic precursor ceus. 3) To detemine the effects of recombinant human bone morphogenetic protein-7 (BMP-7;recombinant human osteogenic protein- 1, rhOP- 1) on fetal rat calvarial cells using parameters of osteogenic differentiation obtained hmthe fkst objective. 4) To relate the perïrnembranous pattem of OPN staining to ceU migration and to examine the potential association of the OPN with components of the CD44 ce11 attachent complex using confocal microscopy and biochemical analyses. The studies carrieci out in accordance with each of these four objectives are organized into corraponding chapters (Chapters 2-5). CHAPTER II

Single Cell Analysis of Intracellular Osteopontin in Osteogenic Cultures of Fetal Rat Calvarial Cens ABSTRACT Osteopontin (OPN), a major component of the bone ma& is expmed at diffèrent stages of bone formation. To detamine possible relationships between OPN expression and stages of osteogenic cell differentiation I perfod single ceil analyses of intracellula. OPN in edy (proliferating), sub-coduent (differentiating) and mature (mineralizxng) cultures of fetal rat calvarial cells using a combination of flow cytometry and confocal microscopy. At each culture stage a high proportion (60 - 98%) of cells were immunoreactive for OPN (OPN+ve). Each of these populations also included a srnail proportion of OPN-ve ceh which were characterized by their small size, low granularity, high proMerative capacity and eatianced osteogenic potentid. The OPN+ve ceiis displayed two distinct patterns of intraceilular immunostaining; a perinuclear distribution typical of secreted proteins and a perirnembrane distriion in which patcha of OPN were concentrated at the celI sudace. Perimernbranous staining predominated in migrant cells, which containeci HO-fold higher levels of OPN than non-migrant ceus as separated in a Boyden chamber. When ceii proHeration was high @ay 2) moa cells were OPN+ve. At alI culture stages the intensity of OPN staining was increased as cells progresseci through the ceU cycle. As celis differentiated and started to fom rnatrix (Days 4 and 6), the mean ce11 expression of OPN was also increased (4-fold), independent of changes in total ce11 protein However, despite the association of OPN with osteogenic cells a high proportion (60%) of fetal skin fibroblasts were also immunoreactive for OPN. The expression of OPN by these ce11 populations was cobedby RT-PCR and a strong correlation was obsmed between the quantitative flow cytometry data and Western blot analysis of ceil extracts in which the high and low phosphorylated isoforms of OPN were observed. These studies therefore, have identified several phenotypes in FRCC cultures that are based on OPN expression: srnall OPN-ve cell populations enriched in osteogenic precursors; differentiating osteogenic cells that synthesize and secrete OPN; and migrating stroma1 cells characterized by a perimembranous OPN staining pattem. INTRODUCTION Mamrnalian strornai celi populations include cells of different lineages (Le. osteoblasts, chondroblasts, firoblasts, myoblasts, and adipocytes) at different stages of differentiation. These cell populations are believed to onginate hm a common progenitor celi (Owen,1988). However, the series of differentiation events that lead to the formation of thae specialized ceU populations are still largely obscure. In osteogenic models, discrete stages in the development and formation of osteoblastic cells have been identified (Owen et al., 1990; Lian and Stein, 1992; Aubin and Heersche, 1992) that correspond to developmental changes dirruig bone formation Ni vivo (Aronow et al., 1990). Associated with these developmentai changes are alterations in the temporal expression of matrix proteins in vivo (Yoon et al., 1989; Chen et al., 1992; 1993) and in vitro (Owen et al., 1990; Lian and Stein, 1992; Ibaraki et al., 1993; Yao et al., 1994). Conceivably several of these rnatrix proteins could be used as osteogenic cell limage markers. One of these proteins, osteopontin (OPN), appears to be highly expressed at two stages of osteogenesis: at an early, proliferative stage and at a Iater stage, subsequent to the initial formation of minefalized bone matrix (Yao et ai., 1994). The early expression is consistent with the immunolocalization of OPN in pre-osteoblastic celis, indicating that this protein could be an early indicator of osteogenic differentiation (Mark et al., 1987). OPN is a glycosylated phosphoprotein exhibiting structural variation through post- translational modifications of the nascent protein (Denhardt and Guo, 1993). Several regions of the OPN molecule are conservai including an RGD site, a thrombin cleavage site, a polyaspartic acid sequence and serine phosphorylation sites (Sodek et al., 1995). The RGD sequence can mediate ce11 attachment and ce11 migration, as well as intracellular signalling through the aJ3, and other htegrins (Yue et al., 1994; Liaw et al., 1995; Miyauchi et al., 1991). The same integrins (Denhardt et al., 1993) and the CD44 receptor (Weber et al., 1996) may also mediate the chernotactic activity of OPN for macrophages and osteoc 1st precursors. The polyaspartic acid sequence appears to mediate OPN binding to hydroxyapatite (Oldberg et al., l986), and phosphate groups may influence hydroxyapatite crystal growth (Hmter & Goldberg, 1993). In bone, OPN has been localized to the osteoid and to the mineralized bone rnatrix and it is also present in mineralized cartilage (Mdet al., 1987; Chen et al., 1990). In remodehg cortical bone, seams of emiched OPN staining have been observed at sites of cernent formation and in lamina Etnitans structwes beneath kgcells (McKee et al., 1993), which may represent early and late events in bone formation (Sodek et al., 1995). However, while OPN is recognized as a prominent bone ma& protein, produced and secreted by osteoblastic celis, the same gene is also expressed by cek in other tissues including kidney and speciahed epitheiia, and by activated lymphocytes, macrophages and transformed cells (Denhardt & Guo, 1993). Although the precise fùnction of the OPN in any of these tissues is unknown, it is evident that OPN can influence cellular and humoral immune responses (Cantor et al., 1995). As OPN has been suggested as a usefid marker for osteoblast differentiation (Aubin et al. 1992), 1 have analyzed OPN expression by osteogenic ce11 populations at different growth stages in Wo. However, since osteogenic cultures contain ceils of different stages of differentiation, and a ngorous knowledge of the ongin and developmental stages of osteoblasts is incomplete, 1 have used a variety of single cell analytical methods to Unprove the resolution of OPN measurements. This study shows that discrete subpopulations of cells cm be identifid based on intracellula.OPN content, that OPN is expressed by fibroblastic ceiis, and that OPN expression is markedly elevated in migrant stroma1 cells. MATERIALS AND METEIODS Ce11 Culture Fetal rat calvarial ceil populations were prepared by enzymatic digestion of calvariae hm21-day-old fetuses of timed-pregnant Wistar rats as describeci previously (Bellows et al., 1986). Cek firom populations II - V, were plated in T-75 flasks and gmwn in a-minimal essential medium (a-MEM)containing 15% heat-inactivateci fetal calf senim @CS) and antibiotics (100 pgM penicih G, 50 pg/d gentamich sulphate, and 0.3 &ml hgizone). Cells were grown at 37°C in a humidified atrnosphere of 95% air/5% CO,. After 24 h incubation, attached cells were washed with PBS to remove non-viable cells and then released with 0.01% trypsin in citrate buffer. CeUs fiom populations II - V were pooled to permit an analysis of the total osteogenic population as well as those fibroblastic cells that are derived hmthe fibrous periosteum. Aliquots were electronically counted (ZM Codter Counter) and replated in T-25 flasks at a density of 7.5~1o4 cells per flask. Culture conditions were identical in all experiments except as outlined below. Cells were grown continuously for a period of 2, 4, 6 and 12 days and the medium changed every 2 to 3 days. Flow Cytomeny Cells were harvested using 2 ml of a proprietory, enzyme-fiee ce11 dissociation buffer (Life Technologies, Burlington, ON, Canada) with Ca"-~&'-fkee PBS, washed in the flask for 30 sec to remove debris and dead cels, and then incubated with the same buf3er for 3 to 4 min. Phase contrast microscopy showed that this method was effective at rernoving al1 cells fiom the culture dish and consistently produced single ce11 suspensions fkom cultures at al1 tirne points. Thus I was confident that al1 cells were included in the analyses. Cells were gently pipetteci several times, transferred to a fkesh tube and incubated with an equal volume of 2% padormaldehyde in Ca"-Mg'-&ee PBS for 30 min and permeabilized with 0.01 % Triton-X to permit staining of inûaceildar proteins with the antibodies. The staining of cells consisted of two labeling steps which included immunocytochemical staining for two bone celI glycoproteins and stauüng with a DNA specific dye for ce11 cycle analysis. Two different antibodies to rat OPN were used in this study; a mouse monoclonal antibody (MPIIIBIOI) was obtained hmthe Deveiopmental Stuclies Hybridoma Bank (Johns Hopkins University, Baltimore, MD under contract hm NICHD). This moue anti-rat OPN antisenun was diluted 12300 in 0.25% BSA in Ca2+- M$-f?ee PBS. A goat polyclonal anti-rat antibody raised against the high-phosphorylated form of rat OPN (Pinero et al., 1995) was obtained fkom W.T. Butler (Houston, TX). This mtiserum was diluted 1:300 in 0.25% BSA in Ca3-~g%eePBS. An affiriity-purifiai rabbit polyclonal antibody against porcine SPARC/osteonectin (ON; Zhang et al., 1986) was dilutecl 1:20 in the same 0.25% BSA solution. After fixation, celis were washed with 0.25% BSA solution, centrfiged and the pellet was incubated with 2 ml of either OPN autibody for 30 min or ON antibody for 1 h at 4OC, followed by 10 min at room temperature (RT; 22OC). The samples were washed again with the BSA solution and pelIeted again. The OPN samples were incubated with FITC-conjugated sheep anti-mouse antiiody diluted 1: 100 in the BSA solution and incubated for 30 min at 4OC. For the rabbit anti-OPN and anti-ON antibodies, cells were incubated with R-phycoerythnn-conjugated (PE) goat anti-rabbit F(ab), hgments diluted 1:20 in the same BSA solution and incubated for lh at 4OC. Stained sarnples were washed with CaL'-M$-fke PBS, pelleteci again and re-niçpended in 0.7 ml of 4',6-diamidino-2-p heny lindole dibyclmc hloride (DAPI; Boehringer, Mannheim, Germany; 1 pg/ml final concentration in 0.1% NP-40). One sample of cells was assessed for total intracellular protein content. These ceus were washed immediately &er para€onnaldehyde fixation (1% final concentration) with Ca"-Me-fiee PBS and re-suspendeci in a mixture of DAeI and sulforhodamine 101 (Texas red; Molecular Probes, Eugene, OR; 20 pg/ml halconcentration). Cells were incubated for 10 min at RT before flow cytometry analysis. Flow cytometry was performed on a FACStar Plus flow cytometer (Becton Dickinson Immunocytotochemistry Systerns, Mountainview, CA) equipped with two argon ion lasers. Two-color excitation was used for analyzing the cells: the 488-nm beam was used for excitation of FITC-conjugated, PE-conjugated second antibodies and sulforhodamine. The 362-nrn beam (U.V.) was used for DAPI. FITC fluorescence was measured at an ernission range of 5 10-530 nm with a band pass filter in the emission path. PE fluorescence was measured at 550-600 m. Tbresholds were set for each analysis using the same celis fixeci and stained with FITC or PE-conjugated antibodies alone. Three color analysis was tried in eariy expaiments and due to inadequate color separation between the two antibodies was abandonecl. The stoichiametry of DNA stahhg by DAPI was verified before each analysis by staining calfthymocyte nuclei as standards. Gating windows were established for the foilowing parameters: FTïC fluorescence; PE fluorescence; sulforhodamine fluorescence; fornard iight scatter (FSC) and side scatter (SSC) were assased only for cells that exhïbited 2N, 4N or intermediate DAPI staining. Light scattering was also computed for ceils either above the detection threshold for FITC or PE, and a second group was assessed for cells below the threshold (i.e. negative staining cells). Four replicate experirnents were conducted and 3x 104cellswere anaiyzed in each sample- Cytospin preparations of ceil suspensions were prepared as described (McCulloch et al., 1991) and analyzed by irnmunofluorescence microscopy for intra and extracellular stahing of OPN. Time-course To study the expression of OPN at different developmental stages, time-course experirnents were conducted based on the three main stages in fetai rat calvarial osteoblast culture development: proliferation, matrix development, and minefalization (Owen et al., 1990). Cultures were studied on day 2, 4, 6 and 12 at the plating densities used in this study. On Day 2, at the peak of proliferation, cultures were sparse and there were many 'H-thymidine labeled ceIls and no cell-to-ce11 contact. On Days 4 and 6 there was a transition between ce11 proliferation and differentiation as show by matrix formation and the appearance of many alkaluie phosphatase-positive cells. At days 4 and day 6, the cultures were approaching confluence, cet1 contacts were extensive, more cuboidal cells were present but there was no multilayering. On day 12 1 observed multilayered cells in discrete foci that became rnineraiized (i.e. bone nodules). Confocal Microscorn> Cells grown on coverslip chamber slides (Nunc, Roskilde, Denmark) were stained for OPN using the same methods as describecl for flow cytometry analysis and examined with a 40X, 1.3 N.A. oil immersion objective with epifluorescent optics and confocal irnaging (Leica). Anal'ysii of lntrucellulaz and Serreted OPN by SDSPAGE Intracellular content of OPN was comparecl to semeted OPN in day 4 and day 6 cultures. Cens in 60-mm dishes were washed twice with 5dof a-MEM containhg 0.5% FCS and re-fed with Sm1 medium. Mer 1 h the conditioned medium was coiiected and replaced with 5ml fkh medium. These steps were repeated twice to collect medium conditioned for 2 h and 3 h. The cells were washed three times with coId PBS and then scraped into 1 ml of 50 mM Tris-HCL 10 mM CaCI, pH 8. The cells were sonicated twice for 10 sec (seming 7; Branson, Danbury, CT) and the supernatant was clarifiecl by centrifügation at 10,000 x g for 7 min. Uther samples nom late mineral stage were prepared using 0.5 M EDTA extract as discribed (Kasugai et al., 1991). SDSRAGE electrophoresis of proteins in the ceii extract and culture media was conducted using gels in Tris-triche buffers as described in detail previously (Yao et al., 1994). Samples of medium and ceil extract were loaded into individual welIs, an equivalent amount of cell extract and medium were also digested with thrombin (5 units of human thrombin; Sigma Chernical Co., St. Louis , MO) to cleave OPN and thereby provide another measure for venfication. Watern Blots Proteins separated by SDS-PAGE were transferred to nitroceildose membranes (0.45 prn pore size) and immunoblotting perfonned using the ECL Western blotting products (Amersha., Life Science, Oakville, ON). Non-specific binding sites were blocked by 3% non-fat dry rnilk in a solution of PBS/O.l% Tween-20 for 1 h. Al1 antibodies were diluted in 0.01% PBSlTween (PBS-T) solution. Primary OPN monoclonal antibody (l:400 dilution) was added for 1 h followed by extensive washings with PBS-T. Horseradish peroxidase-conjugated sheep anti-mouse was added (1:2000 dilution) for an additionai 1 h, followed by washing in PBS-T. Immunoreactive protein was visualized by cherniluminescence detection using reagents accordhg to the manufachuers instructions. Blots were exposed to Kodak autoradiography film. CelZ Migration To determine whether differences in OPN content of cells might relate to their ability to migrate, cells hm6 day osteogenic cultures were separated hto migrant and non-migrant populations using a modifieci Boyden chamber. To obtain mcient ceII numbers for these experiments, migrant and non-migrant celis were separated on large (18

CTI?) polycarbonate membranes (8 ppore size; Poretics Corporation, Livermore, CA) as desmieci previously (Arora and McCuUoch 1996). Briefly, the membranes were treated with 0.01% acetic acid, washed, and coated with a 0.1% w/v coliagen solution. Cells (3x104 were added to the upper chamber and ailowed to attach to the filter in normal growth medium for 2 h. Attachecl celis were thoroughly washed in phosphate-buffered saline (PBS) at 37'C, the chamber was assembled and migration was induced by incubation with fetai bovine senim in the medium of the lower chamber. The cells were incubated at 37'C in a humidikd atmosphere of 95% air/5% CO,. At the end of the assay filters were thoroughly washed with PBS and celis scraped into 1 mi of 50 mM Tris-HCL lOmM CaCL pH 8. Ce11 lysates were collected separately fkom the top and bottom surfaces of the filters and analyzed by immunoblotting for the expression of OPN and actin which was used to normalize for differences in ce11 number. A mouse anti-actin antibody (Sigma, clone# AC-40) was used to detect actin. Previous studies have established that this method effectiveiy normalizes loading of ce11 nurnbers fiom this type of filter (Arora and McCulloch 1996). RT-PCR mRNA analysis To demonstrate OPN gene transcription in different ce11 types and during the different developmental stages of the FRCC cultures, celis were andyzed for the presence of OPN mRNA. Due to the limited number of cells ùi early stage cultures, reverse transcriptase polymerase chah reaction (RT-PCR)was utiked for analyzing OPN expression. The number of cells in T-25 flasks at each of the culture time points was determined with an electronic particle counter and collected. RNA was extracted using the Mini-GT Protocol for preparation of total nucleic acids (Brady et al., 1993). Briefly, 5 pl aliquots of 104 cells were lysed using 100 pl of GT solution (5M guanidine thiocyanate, 0.5% sarkosyl, 25 mM sodium citrate, pH 7.0, 20 mM dithiothrietol). Total nucleic acids were recovered by ethanol precipitation following addition of 50 pl of 7.5M ammonium acetate, 20 pg giycogen as a carier (Boehringer-Mannheim) and 300 pl ethanol. The solution was incubated for 30 min on ice and centrifùged at 4OC for 30 min. at 14000g. The pellet was washed 3 times with 70% ethanol, air-dried and then re-suspendeci in 50 pl cDNA synthesidiysis buffer (52 mM Tns-HCl, PH 8.3, 78 mM KCI, 3.1 mM MgCl,, 0.52% Nonidet P-40 containing 2 U/pl RNAguard (Phannacia). RNA extracteci hmthe osteosarcorna ceIl line ROS 1712.8 was used as a positive control since these ceils express OPN (Wrana et al., 1991). The GeneAmp RNA PCR Kit (Perkin-Elmer Cetus) was used for cDNA synthesis and the PCR amplincation, following the supplien protocol with minor modifications. Random hexamers were used to prime the cDNA synthesis (20 pl ha1 volume) hmthe RNA of 200 cells or from hgof total RNA £iom ROS 17/23 cells. This concentntion of ROS 17/23 RNA was used to approxhate the amount of RNA fkom 200 cells. The reactions were incubated for 10 min at RT to allow random hexarners to anneal? followed by one cycle in a Perkin-Elmer 480 DNA themial cycler as follows: 60 min at 42OC. 5 min at 9g°C, and cooling to 4OC. The cDNA solutions were stored at -20°Cor used immediately for PCR. Serni-nested PCR was perfomed to confirm that the amplifieci product was derived from OPN cDNA. The following primers (Sodek et al. 1995) were used to detect OPN: S-9: 5' CTCKGGTGAAAGTGGCTGA 3'; S-10: 5' TGACCTCAGTCCGTAAGCCA 3'; S-1 1: 5' TGGCTTACGGACT GAGGTCA 3'; S- 12: 5' GACCTCAGAAGATGAACTCT 3'. To minirnize false starts, al1 reactions were assembled and maintained at 4OC until they were loaded into a thermal cycler preheated to 8S°C. In the primary PCR reaction, primers S-9 and S-12, (corresponding to nts 137-156 and 957-1008, respectively in the rat OPN mRNA sequence) were used to ampli@ an 87 1 bp target which incorporates nearly al1 of the coding sequence for rat OPN. Sarnples (20~1)were overlaid with oil and loaded into a preheated thermal cycler. PCR was carried out for 40 cycles under the following conditions: denaturation at 94'~for 1 min, annealing at 57C for 1 min and extension at 72'C for 1 min, halextension at 72OC for 10 min, one cycle. Sarnples were stored at 4OC. In the secondary PCR, 2 pl of a 1 :100 dilution of primary PCR product was added to 23 pl of PCR master mix supplemented with 0.8 mM d.NTPs. The amplification conditions were identical to those used for primary PCR. Primer sets for secondary amplification were S-9 and S-10 (corresponding to nts 503-522) which generates a 385 bp product or S-1WS-12 (corresponding to nts 522-541) which generates a 486 bp product. Amplification products were by electrophoresis in a 2% agarose gel in OSxTBE and visualized with 0.5 pglml ethidium bromide. Stafrstical Analysis Ail experiments were repeated at least three times. Data are expressed as meazl~tSEM hm individual experiments and the data shown in figures were representative of the replicates. The flow-cytometry data consistai of single ce11 analyses. The number of cells in each sample was at least 3x lo4and the fluorescence intensity for OPN stainuig was nomally distributed. From these single ceil analyses of OPN fluorescence I was able to ident.ciifferences between subpopulations as low as 25% with pc 0.001.

RESULTS Expresssion of OPN Ni Osteogenic Culfures Examination of cytospin preparations of rat osteogenic culture ce11 suspensions by immunofluorescence microscopy showed that a large proportion of the cells obtained fiom the different culture periods stained positively for OPN and exhibited a broad range of staining intensity (Fig II 1A). In contrast, cells irnmunostained for a second bone matrix protein, ON, were weakly stained and were observed only in ce11 cultures hmdays 4-12. CeIl preparations stained without the primary antibody were not visible (Fig II 1B) and specific immunostaining for OPN was not detected in cells that were not permeabilized (Fig II. 1C). In spread cells on glass, optical sectioning by confocal rnicroscopy showed that OPN was localized primarily in the central region of the cell. These data indicated that the OPN was primarily intracellular. Stainùig was less intense in regions close to either the dorsal or ventral surfaces (Fig II ID). Spread cells stained Ni situ for OPN could be divided into two distinct phenotypes based on the spatiai distribution of protein (Fig II. IE-H). Perinuclear staining, consistent with localkation to the Golgi apparatus was observed in small (Fig II. 1E) and large (Fig II. IF) cells. The OPN fluorescence was often distributed diffbsely throughout the cytoplasm in the smaller cells, whereas in larger cells intense staining was confhed to the perinuclear region. Perimembrane staining was observed in large polygonal ceils in which OPN was resîrïcted to sites resembluig focal adhesions dong ceil processes (Fig II. 1G) and also within the ce11 body (Fig II. 1H). At earlier stages of culture the perimembrane-staining pattern predominated. Analysis of cells by flow cytometry demonstratecl that the monoclonal antïbody stained cells at least 2 Iogs of fluorescence intensity brighter than cells stained with the second antibody alone (Fig II. 2A). For all culture times at least 60% of ceils were positively stained for OPN (Fig II. 2B). The flow cytometry analyses confirmed the rnicroscopic findings in that there were wide variations of the intracellular OPN staïnïng intensity within cultures (Fig II. 2A) and also at different stages of culture (Fig II. 2C). At day 2 OPN fluorescence was 4-6 -fold lower than the other culture periods exafnifled and staining intensity was highest at day 4. These variations of OPN staini.ug were largely independent of the amount of total intracellular protein measured by sulphorhodamine staining (Fig II. 2D), although both OPN and protein content were both increased on day 4. In cornparison to OPN, ON was not detectable at day 2 and throughout the culture period ON+ve cells were relatively rare and weakly stained (Fig D[. 2C) usuig the antibody described in the Materials and Methods. Relatiomhip between OPN+ve CeZIs and CeZl Cycle Ce11 cycle analyses of DAPI stained cells by flow cytometry (Fig II. 3A) showed that for al1 culture times, the large majority of cells were in G, (Fig II. 4A). From days 2-6, 60-70% of the cells were in G, cells and at day 12 >90% of the cells were in G,. The highest proportion of S-phase and G2, cells were at days 2 and 6, respectively. As cells were aiso stained for OPN, two color fluorescence and bivariate analyses also permitted the simultaneous separation of cells into OPN+ve and OPN-ve cells and assessment of their cycle phase (Fig II. 3B). As indicated above, statistical analyses of plots shown in Fig II. 3B showed that most of the cells at the various culture times were OPN+ve and vimially al1 of the G,cells at days 2 and 6 were stained for OPN (Fig II. 4B). Fig II. 1-Immunofluorescent SWgof Fetal Rat Calvariai Ceils (=Cs) with OPN AntiWes. A -Cytospm preparations of FRCCs stained in suspension with OPN anhibody for subsequent flow cytometry dysis. CeUs stained for OPN show considerable variation of staining intensity witb the popdation. B Absence of staining in cells hcubated with FITC-onjugated 2" antibody alone (negative control). C- Non-permeablized cells show a lack of significant OPN staining (lower than the threshold level recorded for 2d anhidy alone as masured by flow cytometry). D- Consecutive confocal microscopic optical sections (nominal thickness of OSpfiom the dorsal to vend surfaces of a rat calvarial ceU showing that OPN staining intensity is highest in the midde sections (4 to 6). E to H- Confocal micrographs through the mid-section of different cells show phenotypic variations of intraceiiular OPN staining (arrowtieads indicate celi boundaries). OPN staining is diffllsely distriiuted in srnall cek (E) with a perinuclear distribution in Iarger cells (F). Perimembrane distniibution of OPN staining resembling focal adhesions m ceii processes (G) or dong the ceii body 0. At &y 4 there were si@cmt miuctions of the % of OPN+ve celis for al1 phases of the cell cycle and these reductions were equivalent to the reduced proportion of the OPN+ve cells in the whole population (Fig II. 2B). For each time period, OPN content and total protein content increased diiring progression through the cell cycle so that G, cells exhibiteci the lowest OPN and protein content and G,, ceils exhiited the most OPN and protein content (Fig II. 4C, D). However, OPN fluorescence was low for dl cells at day 2 and inmased only slightly with transit through the cell cycle. The discrimination of subpopulations based on OPN staining and ce11 cycle position indicated that there may be time-dependent stages of ce11 maturation in the cultures. To assess this possibility with an alternative approach, I used bivariate analyses of DNA and protein content to determine if there were detectable G, subcornpartments at the different culture times (Fig iI. SA-D). In this context G, cells can be classified into two subcompartments, G,, and G,,. Cells with diploid DNA content and very low protein content are classified as deeply quiescent or G,, cells (Darzyniuewicz et al. 1983), whereas G,, cells are actively cycling. Notably, at day 2 there was a small but distinguishable population of G,, cells, which may represent in part differentiated cells released from the calvarial bone that do not proliferate, whereas at days 4 and 6 al1 cells seemed to be actively cycling. Again, at day 12, G,, cells were detectable, consistent with the differentiation of cells in culture. Reiationship beîween OPN. Ce11 Sire and Cytopilasmic Granularity In cornparison to osteoprogenitor cells, mature osteoblasts are large cells with well developed rough endoplasmic reticulum, Golgi apparatus and lysosomd systems (Ham and Cormack, 1985). 1 used flow cytometric measurements of forward scatter and side scatter to estirnate the relative size and cytoplasmic granularity of OPWve and ON+ve ceIl populations at different stages of the culture development. Cytoplasmic granularity gives insight into the relative numbers of intracellular organelles and vesicles. Throughout the culture time course I noticed the presence of low proportions of very small cells with reduced cytoplasmic granularity that were OPN-ve (Fig II. 64B). -a- OPN fiuorcsccncc

caa O

- - - 2 4 6 12 TIME (days)

Fig II. 2 A - Analysis of the Relationship Between Ce11 Number and intraceiiuiar OPN FIuorescence Sraining Intensity in FRCCs. These analyses, perfomed on cek obtained Corn each rime poinî, are representative of 5 independent replicates of the flow cytometry experirnents, and were used to measure specific OPN fluorescence using fluorescence hteruity signais above the threshold detemhed by 2" antibody staining only. B- Histogram showuig the proportion (%) OPN+ve and ON+ve cells in the whole ce11 population, deterinined by flow cytometry anaIysis of OPN and ON irnmunostained ce11 suspensions. Cells were analyzed at 2,4,6 and 12,days as shown. C-Changes in the inmcellular OPN and ON fluorescence ïntensity (meanse-m.) over the tïme course. D- Temporal changes in mean (*.e.m.) total ceUu1a.r protein content as measured with sulforhodamine fluorescence and flow-cytometry. DNA

DNA

Fi II, 3 Relationship Between Ceii Number, OPN Fluorescence and DNA Content. A- Relationship between ceiï number and DNA content as determmed by flow cytometry of rat caidceli cultures, showing the proportion of ce& in each phase (GJG,;S;G2+d of the ceil cycle as hdicated. B-Bivariate plot of intraceiluiar OPN fluorescence against DNA content Thresholds were estabiished for DNA content just below the G, peak and for OPN stahîng intensity the threshold was estabished accordkig to the nonspecific 2d antiiy staining only as detected by flow cyto=try* TlME (days)

Fig II. 4 Cell Cycle Anaiysis of FRCCs Stained for OPN and Protein. A- Distribution of fetai rat calvariai ceils in each phase of the ceil cycle detennined as shown in Fig II, 3A, for each culture penod. B- Proportion of OPN+ve ce& in each phase of the ceii cycIe as derived fiom experimentai data shown in Fig II. 3B, for each culture period. C- Mean fluorescence intensity of mtraceiluiar OPN in 0PN-i-ve ce& as a tüuction of ceii cycle phase (meauds.e.m). D- Ceil cycle anaiysis of the mean ceii protein content, detected with sulforhodamiue staining and flow cytometry, for the whole ceU population at each of the culture periods (meanks.e.m.). PROTEIN

Fig LI. 5 Bivariate Plots of DNA Content and Protein Content In FRCCs. A to D- Plots of DNA content and protein content €rom &ys 2 (A), 4 (S), 6 (C)and 12 (D) cultures. Day 2(A) and &y 12@) show sigdïcant numbers of non-cycliog subpopulations with low protein content (G,J that are seen as a tail that extends out of the lower Ieft hand rnargin of the c~tosa~h- OPN +ve ON +ve OPN -ve ON-ve

- 700 OPN +ve 600 PI ON +ve B Li 5.0 1 OPN ove C) ON-ve

TIME (days)

Fig II. 6 Flow Cytometry Anaiysis of Sue and Granuiarity of FRCCs. A,B- Forward light scatter (a masure of ceil size/volume changes) and side scatter (a measure of ceii granularity) as measured in fetal rat calvarial cell populations over the time course as indicated. Mean and s.e.n were calculated for the stained (OPN+ve) and unstained (OPN-ve) populations. 1 used the colony assay method as descrî'bed by Beiiows et ai. (1986), to estimate the relative concentration of osteogenic precursor celis in these diffkrent populations. OPN-ve populations containeci a Cfold higher number of bone nodules generateci hmthese cells compared to unsorteci FRCCs hmthe same isolation and plated at the same density. OPN+ve and ON+ve cells tended to be larger ceils with higher values for cytoplasmic granularity. Characterization of OPN+ve Celk As a mal1 but consistent percentage of OPN-ve cells was found in the osteogenic cultures, it was important to determine if some of the OPN-ve cells may represent non- osteogenic fibroblastic cells. As it is not possible to sort out populations of fibroblasts kom the osteogenic cells, OPN staining was deterrnined using pure cultures of fetal rat dermal fibroblasts (FDF). Although the stauiing intensity was reduced compared to the calvarial cells, approxirnately 60% of the FDF were OPN+ve. Consequently, 1 performed sirnilar analyses on adult skin fibroblasts (ADF) to detennine whether the OPN expression was a characteristic of the embryonic cells. No OPN+ve cells were evident in the ADF (Fig II. 7AJ3). As the expression of OPN by normal fibroblastic ceils had not been reported previously 1 investigated the nature of the protein recognized by the antibody using Western blotting. Proteins extracted kom the rat osteogenic cells grown for 4 days and 6 days in culture were analyzed together with conditioned medium collected afier various time intervals in sem-f?ee medium. Two immunoreactive proteins migrating at 72 kDa and 66 kDa were identified; the slower migrahg protein showed the highest ùnmunoreactivity (Fig II. 7C). A sirnilar result was obtained with ce11 extracts f?om the FDFs, while the adult dermal fibroblasts were negative, hdings that were consistent with the flow cytornetry data. Notably, the mobility of these proteins appeared to correspond to the low phosphorylated (LP-OPN)and high phosphorylated (HP-OPN) forms of OPN identified previously nom radiolabeling studies (Sodek et al., 1995). Thus, the faster rnigrating protein was found to CO-migratewith HP-OPNextracted &om mineralized bone formed in culture by the calvarial cells while the slower migrating protein CO-migrated with the LP-OPN secreted into the culture medium. 1 also used a polyc1ona.i antibody raised against the HP-OPN (44 kDa OPN, Rince et al., 1987) which generated comparable dtsto the monoclonal antiody when used for flow cytometry and Westem blot analysis (Fig II. 8A), the only notable différeace being the greater affinity of the polyclonal antibody for the HP-OPN isoform (Fig II. 8B,C). The immunoreactive bands were fùrther characterized as osteopontin isoforms by their susceptibility to thrombin digestion (Fig II. 8B,C) which cleaves rat OPN into hgments of -32 kDa (Senger et al., 1989). To verify that fibroblastic cells as weU as rat osteogenic cells express OPN, as opposed to binding exogenously available OPN, total RNA prepared fiom cells cultureci for 6 days was subjected to RT-PCR using oligonucleotide primers based on the nucleotide sequence of rat OPN. From an initial amplification using primers that encompass the translated region of the OPN mRNA, an 871 bp product was generated in al1 cultures except for adult dermal nbroblasts. To confirm that the arnplified product comesponded to rat OPN mEW4, the primary PCR product was rearnplified using the original 3'-primer and a S'-primer complimentary to an intemal sequence in rat OPN cDNA. The resulting 486 bp PCR product encompasses the carboxy temiinal halfof OPN. Confirmation of OPN mRNA expression in FDF, osteogenic cells and ROS 17/2.8 cells was provided by amplification of the 486 bp fiagrnent (Fig II. 8D). In con- this band was not observed for the ADFs. OPlV Expression and Cell Migration As OPN expression was observed in rapidly proliferating ce11 populations obtained fiom embryonic tissues, the possibility that OPN expression may be related to ce11 migration was investigated. Migrating and non-migrating FRCC ce11 lysates were collected separately kom Boyden charnber filters and were analyzed for OPN expression by Western blotting. Although both migrant and non-migrant cells expressed OPN, mainly as the LP-OPN isoform (Fig II. 9), probing the same membranes for actin to normalize for ciifferences in cell number showed that the migrating cell population contained HO-fold higher levels of OPN. Moreover, immunostaining of the migrating and non-migrating for OPN cells showed a predorninance of the perimembranous staining (Fig II, 1G,H) in the migratory cells as depicted. ' LP-OPN * f '- a HP-OPN 3 mdO

Fig II, 7 Analysis of OPN in Fetal and Adult Rat Fïbrobhts. A- Proportion (%) of OPN+ve ce& in fetal rat calvariai cells at 6 days (RC), confluent cultures of addt dermal fibrobb (ADF)and fetal dermal fibroblasts (FDF). All ce& were at comparable ceil densities when assesseci by flow cytometry. B- Histogram of intracellular OPN fluorescence intensity shows the same relationshxp as the ciifference m %OPN cek (meamts.e.m-). C- Western bIot analysis of cell extracts fiom fetal rat caivariai ceils (RC) at day 6 and hmfetal dermai fibrobIasts (FDF)and adult dermal fibroblasts (ADF).Nodule stage ceil extracts (N) and E-extracts (0.5 M EDTA) of fetal rat calvarial cells at the bone nocide stage. Ali the ceU extracts showed two immunoreactive bands while the E-extract showed ody the slower migrahg HP-OPN band ADF were negative for OPN. Monoclonal Ab. Polyclonal Ab.

B 1 2 3 4 5 6 LP-oPN > T HP-OPN > Fu--

Fig II. 8 Analysis of OPN Expression by Flow Cytomeüy, Western Blotting and RT-PCR A- IntraceUular OPN fluorescence intensity in conml and TGF-BI treated (1 ngiml) cek was detected with a monoclonal anttidy to OPN, as shown in earlier figures (Monoclonal), or by staining with a polyclonal antiiody (Polyclonal) for OPN and analyzed by flow cytometry. B,C- Western blot of duplicate samples of conditioned medium fiom days 2,4,6 and 12 (lanes 1, 2, 4, 5, respectively) and of thrombin digests from days 4 and 12 (lanes 3, 6 respectively). The bIot was probed fim with the monoclonai auti'body which preferentially recognizes the slow migrating, low phosphorylated OPN (LP-OPN).Subsequently the blot was s?npped and reprobed with the polyclonal antiiody (C) which preferentially detects the lower (hi& phosphorylated; HP-OPN) band of OPN. D- RT-PCR analyses of mRNA for OPN. Lanes 1 and 8, ROS 1712.8 ceh; hues 4-7, FRRCs at days 2, 4, 6, and 12 respectively, codbmhg the presence of OPN mRNA; iane 2, feDi1 dexmai fibrobIasts; lane 3, addt dermal fibroblasts. Amplification of a 486 bp fragment con£ïrmed the expression of OPN &A in al1 cek except the addt ddfibrobh (lane 3). Fig II. 9- Western Blot of OPN Rotein in Migrating FRCCs (M) and Fetal Dermal Ce& Compared ta Non-Migrating (S) Cek. CeUs were separated using a modifieci Boyden chamber and the stationary (S) and migmt ceb (M) were smped off the fiiters, Iysed and blotted- Biots were double iabeIed with an anti-actin antiiody to normalize variations of Ioading. Anaiyses were based on triplicates for each celi type. DISCUSSION OPN exhï'bits several features that suggest a potentiaiiy important mie in osteoblast fiuiction, in osteogenesis and in bone remodelling. Firsf OPN is a prominent noncoilagenous protein in the extracellular matrix of bone ceils. Second, the RGD sequence in OPN (Oldberg et al., 1986; Denhardt and Guo, 1993) provida several potential cell regdatory fùnctions that are mediated through the qP,,, integrins (Miyauchi et al., 1991; Yue et al., 1994). Third, polyaspartic acid sequences and phosphate groups rnay mediate binding to mineral (Oldberg et al., 1986; Hunter and Goldberg, 1993). It has also been suggested that OPN may act as a cytokine (Patarca et al, 1993) and ment studies have shown that OPN can mediate chernotaxis and atiachment of monocytic cells through the CD44 receptor (Weber et al., 1996). AU of these putative fiinctions of OPN which can influence osteoblasts relate to its presence in the extracellular ma&. In this study I have assesseci the relationship of intracellular OPN content in single cells to the maturation of osteogenic cultures and to migrahg cells. bh.acelIuIar Osteopontin Immunofluorescence staining of cytospin preparations with and without cell pemeabilization as well as optical sectioning by confocal microscopy confirmed that the OPN detected by the antibody staioing was indeed intracellular. The specificity of the staining was also demonstrated by fluorescence microscopy and flow cytomeûy examination of controls stained without the primary antibody. Also, as remarkably hi& proportions of OPN+ve cells were detected at al1 stages of culture maturation, as well as in ernbryonic fibroblastic cells, 1 used two different antibodies and Western blotting to confirm that only OPN was being recognized. Both antibodies recognized two protein bands, conesponding to the LP-OPN and HP-OPN isofoms of OPN, which were susceptible to thrornbin. Finally, analyses of OPN rnRNA by RT-PCR demonstrated OPN expression in the different ce11 types that paralleled the OPN immunofluorescence and Western blotting data Thus, 1 conclude that the analytical methods and reagents used in this study provided accurate representations of the presence and levels of intracellular OPN. By confocal microscopy 1 identified two discrete patterns of intracellular OPN staining, with some variations apparent in individuai cek. These staining patterns suggested the presence of severai phenotypes which were found at ail stages of osteogenic cultures; but their biological signincance is presently unknown. One patteni, in which perinuclear stalliing was predominant, is typicd of Golgi staining associated with secreted proteins and Likely represents differentiating celis that are fonning an extracellular matrix. The second pattern was strongly reminiscent of staining pattems observed for focal adhesions and was the predominant pattern in migrating cells. As OPN is a ligand for CD44 (Weber et al., 1996) which also interacts with the focal adhesion protein radwn (Tsukita et al., 1994), I suggest that a form of intracefldar OPN may regulate the hction of focal adhesions including the adhesive properties of migrating osteoblartic and other stroma1 cells. In this context, the rat osteogenic cuitures used here contain migrant ceils and, as 1 have demonstrated in this study, migrant celis contain much more OPN than non- migrant cells. Thus, it is conceivable that OPN may regulate the adhaive properties of osteogenic cells that migrate during bone development. That OPN may have fûnctions inside the ce11 is also suggested hmthe demonstration that the OPN content in bone cells is cell-cycle dependent and parallels the content of total ce11 protein. Thus as cells progresseci through the ce11 cycle f?om G, to G,,, OPN content was increased, an observation that indicates that OPN does not behave as a typical secreted protein (Ko et al., 198 1). Conceivably one of these fbctions is to modulate the interaction of focal adhesion proteins with the cytoskeleton and integrins. OPN Expression in Osteogenic Cultures 1 have demonstrated that bone cells are not the only connective tissue cells that express OPN. Although smooth muscle cells have been reported to express OPN (Giachelli et ai.,1995), the expression of OPN by demial fibroblasts has not been reported previously. While the relatively mature adult dermal fibroblasts were found to be OPN-ve, cells that have more "embryonic" characteristics such as transformed cells and fetal dermal fibroblasts also expressed OPN. This finding expands the List of ce11 types that express OPN (Denhardt and Guo, 1993) and points to a common feature of embryonic and metastatic cells that rnay be served in part by OPN: the ability of these cells to rapidly prolifmte and migrate. The presence of ceh of different Iineages in osteogenic cultures complicates studies of osteoblast differetltiation. The hding of OPN expression by skin fibroblasts fiuther confoimds the issue and indicates htOPN expression alone is unlikely to be a useful mark for identification of osteogenic cells. To address this problem 1 have combined cytological approaches (forward and rÏght angle Light scatter) with OPN and ON expression to perform more detailed analyses of the types of cells that are found in maturing osteogenic cultures. These data show that ON is poorly related to osteoblast differentiation but as expected, when ce11 proliferation gives way to ce11 differentiation (days 4 and 6), there were large (4-fold) increases of OPN content. This hding presumably indicates that the more dinerentiated ceus produce large arnounts of OPN for matru< formation at this stage of culture development. Notably, OPN has been show to be produced early in bone formation and has been associateci with the formation of a cernent layer upon which osteoblasts differentiate and form the bone matrix proper (McKee and Nanci, 1995; Sodek et al., 1995). The combined analytical approach of OPN staining and light scatter indicates that there are discrete subpopulations of cells that can be characterized by lack of OPN staining and low cytoplasmic granuiarity. Conceivably, these cells may be precursor cells that are poorly differentiated (Le. low amounts of rough endoplasmic reticulum. lysosomes and Golgi apparatus) and that also do not express OPN. Although I cannot exclude the possibility that these ce11 populations include precursors of other Lineages, the enhanced booe-forming potential of this population shows that they are enriched in osteogenic cells. The presence of small quiescent cells with low cytoplasmic granularity and low total protein content was also identified at the early and late stages of the culture using bivariate analyses of DNA and protein. These ce11 populations could include OPN-ve osteogenic precursors that when stimulated by replaiing in low ce11 density, re-enter the ce11 cycle and contribute to new osteogenic populations. However, the characteristics of this population are also consistent with the presence of quiescent highly differentiated cells that have stopped cychg (Darzynkiewicz et al., 1983). Using a strategy of single ce11 analysis these studies have identifïed several phenotypes based on the expression of OPN in osteogenic bone ce11 populations derived hmfetal calvaria: 1) Small OPN-ve cells with low granularity and high proiiferative capacity that appear to be emiched in osteogenic precursors; 2) OPN+ve cek that exhibit a Shang perinuclea.staining pattern are likeIy differentiating cells thai fom bone matrix; and 3) OPN+ve cells that show migratory characteristics and are characterized by a perimembranous staining pattem. However since OPN is also expressed by fibroblastic ceus, OPN expression done is not an indicator of osteogenic differentiation in cultures of mixed ceil limages. Nevertheles, a combination of light scatter and OPN expression could be used to refine the discrimination of osteogenic ce11 populations as 1 have shown above. Current studies are focussed on the Mercharacterization of the ce11 populations using these rnethods. Characterization of Stroma1 Progenitor Cells Enriched by Flow Cytometry ABSTRACT The progenitors for cells of bone, cartilage, fat and muscle are thought to be derived hmrnesenchymal stem ceils but in spite of extensive study of stromal celI diEerentiation, neither the rnesenchymal stem ceils nor the more committed, tissue- specific progenitors have been well characterized. In this study 1 used flow cytometry to isolate from fetal rat periosteum a population of small, slowly cycling cells with low cytoplasmic granularity (S cells) that display stem cell characterktics. On plating, S cells exhibited a 90% higher labehg index with [3H]-thymidinecompared to unsorteci cells and when grown in culture generated cartilage, adipocyte, and smooth muscle phenotypes, in addition to bone. ûnly the S ce11 population demonstrated extensive self-renewal of cells with osteogenic potential. Electron microscopy showed that S cells have high nuclear:cytoplasmic ratios with large condensed nuclei and a paucity of cytoplasrnic organelles. Freshly-sorted suspensions of immunocytochemically stained S cells did not express differentiation-associated markers such as type 1, II, and III collageos, alkaline phosphatase or osteopontin. However following attachment, S cells became immunopositive for collagens 1, II, III, osteopontin and also for the ce11 surface receptor CD44,which mediates ce11 attachent to hyaluronan and osteopontin. These studies demonstrate that viable osteogenic precursor celIs with the stem ce11 characteristics of self-renewal, high proliferative capacity and multipotentiality can be enriched fiom heterogeneous stromal ce11 populations with simple flow cytometric methods. These cells may be usehl for regeneration of stroma1 tissues. INTRODUCTION The ability of tissues and organs to develop, remodel, regenerate and repair is dependent upon the existence of stem cells that upon division fom more differentiated progeny (Hall and Watt, 1989; Potten and Loeffler, 1990). The existence of stem ceils has been weU-docurnented in the epidennis (Potten, 1976), the intestinai epithelium (Leblond and Cheng, 1976) and the hematopoietic system (Till and McCulloch, 1961). In contrast, evidence of stem cells in mesenchymal tissues is largely indirect(Owen, 1985; Owen and Friedenstein, 1988). In-vivo and in-vitro studies have provided evidence of osteogenic precursor cells in bone marrow and other stroma1 ce11 preparations (Friedenstein, 1976; Gngoriadis et al., 1988; Haynesworth et al., 1992; McCulloch et al., 1991; Van Vlasselaer et al., 1994) but the identity of cells in these tissues and their relationship to cells with classical stem ceil characteristics (Hall and Watt, 1989; Potten and Loeffler, 1990) has yet to be established. Differentiation of mesenchymal cells has been extensively studied in osteogenesis (Aronow et al., 1990; Aubin et al., 1992; Lian and Stein, 1992; Owen et al., 1990). However, the lack of unique markers for osteoprogenitors, and the low estimated frequency of these precursor cells; 0.0005% in bone marrow (Falla et ai., 1993); 0.3% in the fetd rat calvariae (Bellows and Aubin, 1989) has been an impedirnent in the search for osteogenic stem cells. lndeed single ce11 analysis of the phenotypic characteristics of osteogenic cells has indicated that the precursor cells may be heterogeneous (Liu et al., 1994). Nevertheless, attempts have been made to separate osteoprogenitor and osteogenic cells using density centrifugation (Falla et al., 1993) and flow cytometry using cell surface markers (Long et al., 1995; Turksen and Aubin, 1991; Van Vlasselaer et al., 1994). However, successful application of these approaches has been limited by the relatively poor viability of the sorted cells. Further, it is uncertain how closely these cells are related to putative osteogenic stem cells. Indeed, the use of positive selection to isolate progenitor cells would seem to select for relatively differentiated cells which have already begun to express phenotypic markers associated with osteoblastic di fferentiation (e.g. alkaline phosphatase; Turksen and Aubin, 199 1). In a recent study 1 used multi-parametric flow cytornetry analyses of osteopontin (OPN) expression, protein content and ceIl cycle position to identiQ discrete subpopulations of osteogenic cells in fetal rat periosteum at different stages of culture. 1 identifiai at the time of peak proWeration a unique sub-population of small, non cycling OPN-negative cens with low cytoplasmic granulanty and low protein content (Chapter II; Zohar et al., 1997). Since vital sorthg based on cell size and cytoplasmic granularity have been used previously to separate primitive precursors fkom bone marrow hematopoietic cells (Blanc et al., 1991), and to enrich for stem cells for hematopoietic therapy (Verbik et ai., 1995), this approach was used to enrich for a viable sub-population of cells with the characteristics of mesenchymai stem cells. MATERIALS AND METEIODS Cell Culture Fetal rat calvarial celi (FRCC) populations were prepared by five squential enzymatic digestion (14) of calvariae hm 2 1-day-old fetuses of timed-pregnant Wistar rats as described previously (Chapter II). Cells fkom digestions II-V were plated in T-75 fiasks and grown in a-minimal essential medium (a-MEM) containing 15% heat-inactivated fetal bovine sem(FBS) and antiibiotics (100 pg/ml penicillin G, 50 pg/ml gentamicin sulfate, and 0.3 pg/ml hgizone). In some experiments 1 assessed the abiiity of FRCCs and bone marrow stroma1 cells to support hematopoiesis by culturing non-adherent ceils tiom rat or mouse bone mmw flushes in methylcellulose and Iscoves' MDM (Stern Ce11 Technologies, Vancouver, BC; 3x10' cells/mI). Stromal cells were grown at 37OC in a humidified atmosphere of 95% air/5% CO,. Mer 24 h incubation, non-viable cells were washed away with PBS. Cells nom populations II-V were pooled to permit an analysis of the total osteogenic population as well as those fibroblastic cells that are denved korn the fibrous periosteum in these fractions. Aliquots were electronically counted (ZM Coulter Counter; Hialeah, FL) and re-plated in T-75 flash at a density of 2.2Sx10S cells per flask. Culture conditions were identical in dl experiments except as outlined below. Prior to sorting, cells were plated for a period of 2 days. Flow Cytorneby and Ce11 sorthg Attached cells were harvested using 5 ml of 0.01% trypsin in citrate buf5er and cells from five T-75 flasks (-4x106 cells) were re-suspended in 2 ml a-MEM(phenol red- fiee, to eliminate arte factual fluorescence during flow cytomeûy) containing 15% filtered FBS and 10% antibiotics. Sorting was performed on a FACStar Plus flow cytometer (Becton Dickinson Immunocytochemistry Systems, Mountainview, CA) equipped with an argon ion laser (Coherent Innova 70) operating at 250 mW beam power. Gating windows were established for forward light scatter (FSC) and side scatter (SSC). Particles with an average size c5pm (detennined by running standard size beads) were excluded. Two major subpopulations were sorted: cells with the lowest 15% FSC and lowest 15% SSC in the population (S cells) and the cells with the highest 15% FSC and highest 15% SSC (L cells). Three 0th- populations were also collected: 1) FRCCs cells were passed without sorting through the flow cytometer as a control group to evaluate the effect of passage through the flow cytometer on osteogenesis in-vitro and other functional assays; 2) ceils remahhg der sorthg the two main groups (S. L), were designated as S-ve and L-ve respectively. Ail groups were collected in glass tubes containing 3 ml a-MEM with 15% FBS and 10% antibiotics. Ce11 VitaZiîy To assess ce11 vitality as a result of passage through the flow cytometer and sorting, sorted cells were plated overnight into 8-well chamber slides with eight replicates (Nunc, Roskilde, Denmark; n=8 replicates) at a density of 1x10~cells per well. One slide was plated at the same density with unsorted FRCC. Slides were washed with PBS to remove non-viable cells and debns and fixed. Cells were washed, permeabilized with 0.0 1% Triton-X to permit intracellular staining and incubated for 10 min. at 4°C with 4',6-diamidino-2-phenylindole dihydrochloride (DAPI; Boehringer, Mannheim, Gemany; 1 pgM final concentration in 0.1 % NP-40).Slides were andysed by immunofluorescence microscopy. The mean number of cells and standard errors of the means per microscopic field area (25x objective) were computed. Statistical analysis of computed p values for these data were determined using analysis of variance and Tukey's test. OPN mRNA Cells were analyzed for the presence of OPN mRNA by the reverse transcriptase polymerase chain reaction (RT-PCR) as described (Chapter II; Zohar et al., 1997). RNA was extracted using the Mini-GT Protocol for preparation of total nucleic acids (Brady and Iscove, 1993). The GeneAmp RNA PCR Kit (Perkin-Elmer Cetus) was used for cDNA synthesis and PCR amplification following the supplier's protocol with minor modifications. Random hexarners were used to prime the cDNA synthesis (20 pl final volume) from the RNA of 200 cells as described ( Chapter II; Zohar et al., 1997). Semi-nested PCR was performed to confirm that the amplified product was deriveci nom OPN cDNA. Amplification products were analyzed by electrophoresis in a 2% agarose gel in OSxTBE and visualized with 0.5 pg/ml ethidium bromide. Aïkaiine Phosphatase ActMty and Cell Prolifration Sorted cells were plated into 8 well chamber slides (Nunc, Roskilde, Denmark; n=4 replicates) at a plating density of 1xlV ceus per well and were compared to unsorted FRCCs plated at the same density. Cultures were analyzed on day 1, 2,4, 6, 8, 10. Due to the Limited numbers of cells in the sorted subpopulations, only FRCCs were analyzed on day 12. To assess the proportion of proliferating cells, wells were incubated with 'H-thymidine (1 pCi/mI) for 3h before the termination of the culture. Slides were fuced, stained for AP activity, and prepared for radioautography with NTB-2 liquid emulsion (Kodak, Rochester, NY) as described (McCulloch, 1986). The following populations of cells were counted: 'H-thymidine labeled cells (> 4 silver grains per nucleus), AP positive cells, and cells with both 'H-thymidine labeling and AP staining. Al1 cells were counted in the same microscopie field area (40x objective; triplicate fields for each culture). The mean labeling indices of cells and standard errors of the means were computed. Lzmiting Dilution An dysis Cells were plated in 96-well plates at dilutions of 5, 25, 50, 100, 300 and 600 cells per weI1. Two replicate plates (192 wells) were obtained for each dilution. Cells were grown continuously for 24 days and the medium was changed every 2 to 3 days. Ascorbic acid (50 pg/ml) and 10 mM sodium P-glycerophosphate (Sigma) were added at confluence. Mer 24 days wells were fixed ovemight in neutral buffered formalin and stained with von Kossa's reagent. The fiaction of wells without bone nodules (F,; non-responsive wells) was calculated for each plating density. This hction was plotted against the number of cells plated per well for each one of the subpopulations. A linear regression was fitted with 95% confidence lunits and correlation coefficients were calculated. Application of the Poisson distribution (Lefkovits and Waldmann, 1979) permitted estimation of the number of osteoprogenitor cells in each of the groups which upon division was capable of forming bone nodules. Cdony Assays For assessment of osteogenic capacity at relatively high cell densities, sorted cells were plated in 35-mm dishes at 3x104 cells per dish. Two sets of dishes were prepared, either untreated (plastic) or pre-coated with a thin fiIm of collagen type 1 (Vitrogen; Celtrix, Santa Clara) following the supplier's protocol. Cultures were subsequently augmented with 50 pghl ascorbic acid and 10 mM sodium P- glycerophosphate (Sigma) at confluence. The cultures were terminated on day 16, fixed overnight in neutral buffered formalin, and stained with Von Kossa's reagent (Von Kossa, 1901). The number of bone nodules was counted in each plastic dish. Triplicate fields (10x objective) for each culture were counted in triplicate dishes. The mean nurnber of bone nodules and standard deviations were calculated for each group. The mean area of bone nodule was assessed by image andysis (R&M Biornetrics, Nashville, TN) and expressed as pn2;thxty replicate nodules were rneasured for each group (10x objective). The mean nodule size and standard deviations were calculated and differences were evaiuated using analysis of variance. Collagen-coated dishes exhibited diffise patterns of mineralkation which precluded enumeration of bone nodules. Self-Renewal Capacity To assess the self-renewal capacity, cells fiom the two main sorted subpopulations (S, L), as well as FRCCs passed through the flow cytometer without sorting, were plated in T-25 flasks at a plating density of 7.5x104 cells per flasks and grown for 12 days (early mineralization stage (Zohar et al., 1997)). Non-viable cells and debris were removed by washing twice with PBS and attached cells were harvested with trypsin in citrate buffer. Visud inspection of cultures following trypsinization confirmed the complete removal of al1 cells. Aliquots were counted electronically and prepared for sorting as described above. initially, cells were re- sorted into 3 subpopulations (parental FRCC,; S,,L$ using the same criteria as the kt sort. Subsequent sorts used expanded windows for the L subpopulation. Cells were seeded into 12-well chamber plates at a plating density of 1.5x104 cells per well and supplemented with ascorbic acid (50 @ml) and 10 mM sodium P-glycerophosphate at confluence. Cultures were termbted der 16 days of culture, stained with von Kossa's reagent and analyzed for bone nodule formation. The mean and standard deviation of the number of bone nodules were detennined nom duplicate fields (10x objective) counted for each culture in 6 replicate dishes. Electron Microscopy Transmission electron microscopy was performed to assess the morphological characteristics of the subpopulations sorted by flow cytometry. Sorted subpopulations were either fked in suspensions (cytospin) or plated over-night, and then ked with 2.5% glutaraldehyde in 0.1M sodium cacodylate buffer, pH 7.3, post-fixed with osmium tetroxide, dehydrated in an ethanol series and embedded in spurr epoxy resin. Thin sections (70 nm) were stained with uranyl acetate and lead citrate and exarnined under an electron microscope. Immunostaining The two sorted subpopulations (S and L) were charactenzed by antibody staining and the staining patterns were compared with unsorted FRCC. Two sets of stainuig procedures were used. In the fmt procedure, 1.Sx104 cells were sorted by flow cytometry, collected in tubes, fixed immediately, stained in suspension and cytospin preparations prepared as described (McCulloch et al., 1991). In the second procedure, sorted cells were plated in 8 well chamber slides (Nunc, Denrnark) at a plating density of 2x10~cells per well. Cells were grown overnight and washed twice before fixation. Replicate wells (n=4) were obtained for each subculture. Staining procedures were identical in the two procedures and were done simultaneously. Antibodies to collagen 1, II, and III were used as described previously(Bel1ows et al., 1986; Wang et al., 1980). Mouse anti-rat OPN monoclonal antibody (MPmB 101) was obtained fkom the Developmental Studies Hybridoma Bank (Johns Hopkins University, Baltimore, MD under contract nom NICHD). htracellular staining of OPN by this antibody was characterized previously (Chapter II; Zohar et al., 1997). Biotin-conjugated mouse anti-rat CD44 (Pgp- 1, H-CAM; Clone 0x49; Pharmingen, San Diego, CA) was used to stain cells, as OPN and hyaluronan, which is expressed early in development, are important ligands for CD44 (Weber et al., 1996). Cells immunostained for collagens type 1and II were fked with methanol at -20°C for 15 min. Samples irnmunostained for collagen III, OPN, CD44 were fixed with 2% paraformaldehyde in Ca2+-Mc-fkePBS for 30 min at 4°C. Washing and dilution of alI antibodies was in 0.25% BSA in CaZ'-MC-fke PBS except as outlined below. After two washes ceiis were incubated with the folIowing diluted antibodies: an affinity purified sheep anti-rat collagen 1 (1:20) was incubated for lh at 4OC; an af3k.i~purified rabbit anti-rat collagen II (MO) was incubated for lh at 4°C; an af-finity purified sheep anti-pig collagen III (1:20) was incubated for lh at 4OC; OPN (1 2300) and CD44 (1:200) were both incubated for 45 min at 4°C followed by a 10 min incubation at 22OC. Al1 samples were washed twice again with the BSA solution. Collagen type 1 and III stained samples were incubated with FITCconjugated rabbit anti-sheep antibody diluted 150 in the BSA solution and incubated for lh at 4OC. Collagen II stained samples were incubated with FITC-conjugated goat anti-rabbit F(ab)2 hgments diluted 1:20 in the same BSA solution and incubated for lh at 4OC; OPN samples were hcubated with FITC-conjugated sheep anti-mouse antibody diluted I:100 in the BSA solution and incubated for 30 min at 4OC; CD44 stained samples were incubated with streptavidin FITC-conjugated antibody diluted 150 in the BSA soIution and incubated for Lh at 4OC. Stained samples were washed with Ca2+-~g-f?eePBS, and counterstained with DAPI for 10 min at 4OC. Slides were washed twice again with Ca2+-Mg-fieePBS and cytospins of the ce11 suspension were prepared. Slides were coverslipped with Pemount (Fisher, Toronto, ON, Canada) and analyzed by irnrnunofluorescence microscopy. DAPI fluorescence was used to ensure the presence of cells and then cells were examined for specific proteins with the FITC-labeled reagents. FITC fluorescence was graded as: none, low, medium, and high. The relative percentage of stained cells was estimated from counts of the relative number of FITC positive cells divided by the number of DAPI nuclei. RESULTS 1 assesseci whether FRCCs could support hematopoiesis, thereby indicating their potential as stromal cells- Non-adherent hematopoietic cells from either rat or mouse femoral bone marrow flushes did not fom colonies when plated in methylcellulose, Iscoves' MDM and 2% FBS. However when incubated with growth factors (3 U/ml , 10 nghl mrIL-3, 10 nghi ML-6 and 50 ng/ml mr , al1 nom Stem Cell Technologies), colony foming units (CFC-C) were observed (CFC-C-4%3.9/35mm dish). Similarly, when celis were incubated with Iscoves' MDM and 2% FBS and plated on feeder celi layers of either high density rat bone marrow stromd cells (CFC-C -57&4.6/35mm dish) or on high density FRCCs (CFC-C -5&3.4S/3~mmdish), there was colony developrnent and with no statistically different colony counts compared to the number of colonies in cultures supplemented with hematopoietic growth factors. 1 conclude that FRCCs are competent stromal cells. CeZZ Sorting Cells isolated from fetal calvariae were sorted following culture for 2 days, a time of maximal proliferative activity in the FRCCs cultures and when conditions for e~chingfor S ceils was optimal. As determined fiom previous studies (Zohar et al., 1997), S cells were charactenzed on the basis of low fornard angle light scatter, low cytoplasmic granularity, low protein content and e~chmentin G, and S phases of the ce11 cycle. A typical profile of FRCCs sorted on the basis of light scatter is shown in Fig III. 1A and is representative of 7 separate sorting expenments that were conducted. Ce11 counts showed that both the S and L ce11 populations compnsed 6-1 1% of the total number of fkctionated cells. Analysis of cells that attached following overnight plating (Fig III. 1B) showed that, in cornparison to unsorted FRCCs, passage of cells through the flow cytometer reduced FRCCs vitality only slightly (pN.2). However, the L and L-ve subpopuIations Fig III. 1- Separation of FRCCs by Flow Cytomeûy. A- A representative flow cytometry plot of FRCCs separated according to light scatter characteristics reflecting cytoplasmic granuiarity and cell size. Seven independent replicates of the sorting procedure were used to isolate the S (lowest 15% FSC and lowest 15% SSC) and L (highest 15% FSC and highest 15% SSC) sub-populations. This cytogram shows debris close to the origin but particles smailer than 5 pn were exchded from the sort. B- Histograrn showing the number of sorted cells (black bars; mean * s.e.m.) in each popdation that attached to glas afler overnight plating compared to re-pIated FRCCs fiom the same cultures (hatched bar). Counts were done on DAPEstallied ce& and expressed as number per 100 ceiis p!ated. C- Andysis of OPN mRNA expression in sorted cells rneasured by RT-PCR. Samples were &O amplified for B-actin venfying the presence of RNA in dI sorted samples. Lane 1-5, S cek; FRCCs; L cells; S-ve ceh; L-ve celis. Firs~amplification of an 871 bp fragment, encornpassing rnost of the OPN sequence, was carried out using total RNA exîracted fiom the celis (left side). This was followed by amplification of a 486 bp fiagrnent using the 871 hgment as template (right side) which confimied OPN expression in ali populations except the S population. 68 showed -20-25% loss of vitality @<0.05) compared to unsorted FRCC, whereas the S subpopulation exhibited a 40% loss of vitality (pe0.05) which was a significantly higher loss than observed for al1 the other populations wO.05). The lower viability of S cells rnay in part reflect the inclusion of srnall, apoptotic cells which were included in the sorthg window although 1 note that these cells can be readily deleted korn the sort by the use of pulse processing software (Becton Dickinson). To ver@ that the S subpopulation was OPN-ve, RNA prepared fkom the sorted subpopulations (200 propidium iodide-negative cells were used for each preparation) was subjected to RT-PCR using primers based on the rat OPN sequence as described in Chapter II. (Zohar et al., 1997a). Initial amplification of the 871 bp sequence of the translated OPN mRNA generated a positive response in al1 the sorted subpopulations except the S cells which were negative in four replicates and weakly positive in one replicate. When the primary PCR product was re-amplified using nested primers corresponding to the carboxy-terminal half of OPN, the resulting 486 bp product confirmed the presence of OPN mRNA in al1 the subpopulations except S cells which were negative in al1 replicates (Fig III. 1C). Alkaline Phosphatase Activify and Prolifrative Capacity The 'H-thymidine labeling index (Fig III. 2A) for the S subpopulation was the highest of ail the subpopulations in the fkst 48 h of culture. In the fist 24 h of culture the S subpopulation exhibited a 90% higher labeling index than the unsorted FRCCs and >100% more than the L subpopulation. S ce11 nuclei were heavily labeled with silver grains (> 20 grauis/nucleus) whereas the other subpopulations exhibited only 5- 12 grains for labeled cells. The hvo sorted subpopulations (S, L) showed decreased proliferation during the matrix formation phase of the culture (days 46) while FRCCs rnaintained a relatively constant % of labeled cells. As cultures multilayered and approached minerakation (days 8-12), there was another increase in the percentage of labeled cells for al1 groups. Although at day 8 there was no difference between the subpopulations, the S subpopulation at day 10 exhibited 3-fold more labeled cells than the other two groups. Alkaline phosphatase (AP) activity (Fig III. 2B), used as a marker of osteublastic differentiation, was detected in -35% of cells in the FRCCs and L groups during the first 24 h of culture whereas S cells did not dernonstrate any detectable AP activity over this time period AU groups showed increased proportions of AP+ve cells during the matrix formation phase in which the highest proportion were ia the S and L groups (70% of the cells) and 60% of the cells in the FRCC. The L cells exhibited high proportions of AP+ve cells until day 8 when there was a decrease to only 30% of the cells. A simila. decline was observed in the FRCCs on day 10 and these two groups showed increased proportions of AP cells by 545% as they approached mineralization. The S population did not lose the high proportion of AP+ve cells acquired during culture: at least 60% of the cells on day 6 were AP positive. The same fields were analyzed for cells exhibiting dual labeling for ['Hl- thymidine and AP, a measure of the proportion of transit amplifjing cells. In the £kt 4 days only cultures of the parental FRCCs and L cells showed dual labeled cells (Fig m. 2C). The peak proportion for the L population was on day 2 when 40% of the cells in the culture were double labeled whereas only 23% were double labeled for the FRCC. The S group did not show dual labeled cells until day 6 when dl groups exhibited similar proportions of dual labeled cells (20-30% of the cells). Al1 groups showed an increase in the proportion of dual labeled cells as they approached mineralization: 60% of the cells in the S group compared to 15-20% in the FRCCs and L groups were double labeled.

Limiting dilution analysis was used to assess ce11 CO-operativityin bone nodule formation and the relative proportions of transit amplimg cells that could divide in culture and produce bone nodules (Bellows and Aubin, 1989). The analyses showed a linear relationship between the number of cells plated per well and the fiaction of non- responsive wells. The lowest R2value for al1 groups was 0.83 in the FRCCs (Fig III. 3A), demonstrating a strong linear correlation and single hit kinetics, as evaluated by counting of von-Kossa stained bone nodules. 0.0 O 2 4 6 8 101214 DAY

Fig III. 2- Anaiysis of Ceii Proliferation and Alkaline Phosphatase Expression. A- The proportion of 'H-thymidiue-labeled cells at difTerent stages of cellular differentiation was determined (mean labeling index + s.e.rn.) following a 3 h pulse of 'H-thymidine to detect proIiferating cek. B- The proportion of ceus expressing alkaline phosphatase (AP index) at the same time points anaiyzed for cellular proliferation was detemiined by staining ceils with Naphthol AS Phosphate substrate containing Fast Blue BB sa16 and the proportion of blue stained ceIls was counted (me= index I s.e.m.). C- Proportion of ceUs exhibiting both labeling for [)HI-thymidine and AP staining (mean * s.e.m). Thus the progenitor cells that divided to form nodules did not require assistance fiom other cell types. Based on the single event theory (Lefkovits and Waldmann, 1979) the proportion of amplifying osteoprogenitors was estimated for each of the subpopulations. By calculating the probability of no response at F,=û.37, 1 estimated that the proportion of these progenitors in the FRCCs was 1:620 cells, 3-fold less than the S subpopulatioo which I estimated as 1:202 (Fig III. 3B) or -1 :120 when adjusted for ce11 viability (Fig III. 1B). The L group showed 3-fold lower numbers in relation to FRCCs (Fig III. 3C; 1:1750) which was almost 9-fold less than S cells. The S-ve subpopuIation contained more progenitors than L cells (Fig m. 3D; 1:968) but this was still4-5 fold less than the S sub-population. Thus over the time period of culture, al1 populations were capable of generating transit amplimg ceLls for the osteogenic lineage but the relative fkequency of these cells was much higher in the S population. Osteogenic Capacity of Sorted Subpopulations I plated cells on either tissue culture plastic or on collagen-coated plastic as earlier work has shown that collagen innuences the development and maintenance of the osteoblastic phenotype in primary and passaged rat calvarial osteoblasts (Lynch et al., 1995). Within 24 h following flow cytornetry, al1 sorted subpopulations attached to plastic or collagen-coated dishes. On the basis of phase contrast microscopy and staining with oil red, alcian blue and toluidine blue, there was the morphological appearance over tirne of fat cells, chondrocytes and smooth muscle cells (see below) in addition to the presence of osteogenic cells which are the focus of this study. On both collagen and plastic substrats, cell proli feration was most rapid in the S group. Indeed, only the S and L-ve populations had reached confluence on collagen gels by day 13, at which tirne a difie pattern of mineralization was evident. In two of four replicate experiments the FRCCs, S-ve and L subpopulations showed only Limited mineralization after 20 days on coilagen gels and never to the same degree as observed in the S and L-ve populations. S cells grown on plastic formed rapidly mineralizing bone nodules Number of plated ceiis

Fig III. 3- Limiting Dilution Analyses of Bone Nodule-forming Capacity. Linear regression analysis was performed to show the relationship between the fraction of the non-responsive wells against pfated ceii number for the different sorted cell populations (B- D) and the parent FRCCs population (A). Results are expressed as mean I 95% confidence limits. R is the correlation coefficient- F,,,, is an estirnate of the mean number of cek that would inchde one progenitor ce& based on application of the Poisson distriiution. by day 9, as observed by phase-contrast microscopy. in contrast, mineralized bone nodules were not evident in the L-ve and other subpopulations before day 12. However, on day 13 all groups had produced bone nodules except the L population which formed smd nodules 3-4 days later. Notably, bone nodules fomed by the S cells were significantly larger than those forxned by the other populations (see below). Staining of day 16 cultures with Von Kossa's reagent (Fig III. 4A) confirmed the observations by phase contrast microscopy. Anaiysis of bone nodule-fonning capacity of different subpopulations showed that in collagen-coated dishes, most of the dish in S and L-ve subpopulations was covered with rnineralized tissue. There were small foci of mineralization in the S-ve group but no bone nodules in the L group or FRCC. Quantification of bone nodules on plastic dishes (Fig III. 4B) revealed Cfold higher numbers of noddes in the S group than the FRCCs population and 10-fold larger size of individual bone nodules @<0.001). In contrast, the number of nodules in the L group was 3-fold lower ihan the FRCCs population which was sùnilar to the S- ve subpopulation, and 2-fold lower than the L-ve subpopulation. There were no significant differences in the size of nodules produced by the parental FRCCs and L cells while the nodules produced by S-ve cells were Cfold larger than FRCCs and bone nodules produced by L-ve cells were 6-fold larger than FRCCs @<0.001). Serf-Renewul Capacity As counts of bone nodules showed that dl cultures contained measurable numbers of ampiifjhg osteoprogenitor cells albeit at different proportions, I conducted a more rigourous assay to estirnate the self-renewal capacity of more primitive, undifferentiated cells. After 12 days of growth, S and L cells and FRCCs were individually subjected to a second round of flow cytometry to produce S,, L, and parental (unsorted) FRCC, populations of re-sorted cells. Notably, on passage through the flow cytometer, 43% of S-cells retained the same forward scatter and side scatter characteristics of the initially sorted population. In contrast, none of the L-cells exhibited the cytological characteristics of S-cells. Consequently 1 expanded the sorting windows for the L, population to include those cells with the highest 50% side scatter and highest 50% fonnard scatter. . , ,- - . * .-' . .- - - S..

Fig Ln. 4- Analysis of Bone Nodule Formation. A- Photograpb of &y 16 cultures stained by Von Kossa to demonstrate mineralization. Ail subpopulations exhibit mineralization when grown on plastic dishes p) but S and L-ve ceus exhibit Iarger numbers of bone nodules. Mineralization is observed only for S and L- ve cefi when grown on collagen- coated (C) dishes- B- Bone nodde number and size (p)detennined after Von Kossa staining of the day 16 cultures grown on plastic dishes (mean î s.e.m.). Bone nodules were counted in three fields in tripkate dishes for each group. After plating, the Scell populations hmall pups were confluent by day 7-8. Phase contrast microscopy of S, cells showed a progressive increase in ce11 numbers and foci of bone nodule formation for each sorted population. However, L, and FRCC, groups exhibited decreased ce11 dençity hm day 8 onwards. L, cells showed ce11 degeneration, as indicated by enlargement of ceLl bodies, an increased number of cytoplasmic vacuoles and detachment of cell processes. By day 14 there were few cells remainiog in the L, group with somewhat higher ce11 numbers in the FRCC,. Von Kossa staining of day 16 cultures showed abundant bone nodule formation in cells nom the S, group but only barely detectable numbers of bone nodules were observed in the FRCC, and even lower numbers in the other subpopulations. The mean bone nodules counts per field were: FRCC,- 1. l* 1 .O7;L,- 0.0; Ss-54.5I5.02. Pluripotentiaiity In S-ce11 cultures plated at low density (1000 cells per 100 mm diameter dish; 45 days growth), isolated colonies were marked and followed over tirne. From serial morphological observation 1 detennined that the colonies arose fkom single cells (Fig m.5A). Many of the colonies stained positively for oil red (a rnarker of adipocytes), for alcian blue (a marker of cartilage) and for von Kossa' reagent (a marker for osteogenesis; Fig III. SB-D). In some colonies denved nom S-ce11 cultures, stained cells of a single phenotype were colocalized with cells in the same cluster that were of other p henotypes (Le. fat/cartilage or bondfat). In contrast, stained colonies were either absent or were very rare in L-ce11 cultures. Ultrastmctural Charactenzation Electron microscopy of the sorted cells venfied the flow cytometric measurernent of size and cytoplasmic granularity which was used for sorting in this study (Fig m.6A- F). L cells exhibited the largest size and most well-developed endoplasmic reticulum and vacuolar apparatus while S cells were the mallest and least developed in terms of cytoplasmic structure. Higher magnification showed that S cells had a high nuclear/cytoplasmic ratio, condensed nucleus and low arnount of cytoplasmic organelles (Fig III. 6F). L cells exhibited relatively mail nuclei and a cytoplasrn rich in organelles. Fig LII. 5- Evidence for Pluripotentiality by Formation of Multiple CeU Types at Clonai Celf Densities. Sorted S-ceils were plated at ver- low density (1000 ceW100 mm diameter plate) and cdtured for 45 days (A) X4. Note the discrete, isolated colonies distniuted throughout the dish, Colonies were marked, observed over time and found to arise fiom single ce&. Colonies were stained for cartilage by alcian blue (B), for adipocytes by oil red (C) and for bone by Von Kossa's reagent @). Figs. B-D-X400. Fg m. 6.- Electron Microscopy of Sorted Ceil Populations. Ceiis in suspension were examined at low magnification (~1200)to estimate relative ceil size (&Cs),and at high magnification (~8400)to show detaii in nuclei @,ID$). A,B- FRCCs cek; Ca- L ceils; E,F- S ce&. L ceils (C) have the largest diameter ( 14-25 pm) and the S cek (E) the destdiameter (6- 12 p). At high mapfication large condensed nuclei are evident in S celis with a smd number of intraceiiuia. organelles, whereas L ceh (D) exhibit a high number of cytoplasmic organelles and have a more dispersed nucleus with a convoluted nuclear membrane. Sorted - Cytospin Plated over night Protein detected SlLIISIL

Ostcopon tin Cotlagcn-1 Cotlagcn- II Coliagcn- III CD-44

Table III. 1- Immunostaining of ceils that were sorted and analyzed (Sorted-Cytospin) or sorted and plated overaight fiom S and L cell subp~pulationsand sîained with various aatiies. Fluorescence intensity give an estimate of level of expression: - none, + low, ++ medium, * high, * veIy hi&; (%) indicates percentage of cells expressing the specific phenotypic rnarker, 100 cells were examined in triplkate fields in 8 wek analyzed for each group. DAPI agwas used to show presence of viable cells in each population; irrelevant antibody and 2nd Ab done was used to show the absence of non- specifrc staining. Higher magnincation showed that S ceils had a high nuclear/cytoplasmic ratio, condensed nucleus and low amount of cytoplasmic organelles. L ceils exhibited relatively small nuclei and a cytoplasm rich in organelles. Immunofuorescence of Cytospins and Spread Cells Tmmunocytochernical anaiysis of cytospin preparations showed large numbers of DAPI-stained nuclei for al1 preparations. S cells did not stain for OPN (Table III. 1). FRCCs exhibited intraceilular OPN staining in -80% of the cells and staining intensity ranged nom low to hi&; 60% of the L cells were stained for OPN at medium intensity. High intensity collagen I staining was detected in 70% of the FRCCs while low to medium stahing was seen in 60%of the L cells. Medium intensity collagen II stainuig was detected in more than 30% of the FRCCs and 70% of the L cells showed medium-high intensity staining for type II collagen. None of the S cells were immuflopositive for collagens 1 or II. Collagen III stalliing was seen in only a few S and FRCCs cells (< 5%) with low intensity while 20% of the L cells exhibited low to medium collagen-III staining. lrnmunoreactivity for CD44 was not observed in any of the groups, possibly because of alteration of its extracellular compooent due to trypsinization and sorting trauma. Analysis of sorted cells plated ovemight demonstrated large numbers of attached cells stained with DAPI. High intensity intracellular OPN staining was observed in most of the plated S cells and bright staining was also observed in 70% of the FRCCs and the L cells (Table m. 1). All groups exhibited a perimembranous, focal adhesion type of OPN staining (Zohar et al., 1997). Larger intracellular clusters of OPN staining were observed in the L ceils. High intensity but difise intracellular staining for collagen 1 was observed in 40% of S cells and >60% of the FRCCs and L cells. High intensity diffuse intracellular staining for collagen II was observed in 40% of S cells and FRCCs and 80% of the L cells. Bright stainuig for collagen III was observed in 25% of the S cells while 80% of the FRCCs cells showed medium intensity staining and 40% of the L cells showed low-medium intensity. High intensity surfacastainhg for CD44 was observed in 40% of S cells and medium staining for CD44 was observed in 40%of FRCCs and L cells. DISCUSSION Stem Cell Enrichment Strornal cells derived ftom bone and other mesenchymal tissues comprise a heterogeneous population that include cells with high proliferative capacity and muitipotentiality, indicative of the presence of stem cells (Owen and Friedenstein, 1988). However, pnor to my study, cells with the classical features of stem cells have not been clearly identified in ceil populations derived from stromal tissues and only limited progress has been made in isoIating these cells (Van Vlasselaer et al., 1994). 1 have shown here that sorting cells on the basis of size and cytoplasmic granularity enriches for a population of slowly cycling cells that do not express diffkrentiation associated markers and which upon plating develops high proliferative capacity, multipotentiality and capacity for self-renewal. In contrast, the remaining S-ve and L populations were depleted of stem cells as demonstrated by their lack of self-renewal capacity, their limited proliferative capacity and their reduced ability to fom bone nodules. As these populations contained abundant proportions of cells that were stained for alkaline phosphatase and were labeled with thymidine, they likely comprise transit arnplifjmg cells (Potten and Loeffler, 1990). Some of these putative arnplifying cells entered the osteogenic lineage and were therefore capable of producing bone nodules; but they exhibited almost no self-renewal capacity. Since recovery of viable cells following selection and isolation is problematic (Turksen and Aubin, 1991; Van Vlasselaer et al., 1994), 1 used FRCCs primary cultures as a mode1 since this system contains a relatively high proportion of osteoprogenitor cells (Bellows and Aubin, 1989) and stem cells are likely to be present in the periosteal tissue surrounding bone (Owen, 1985). Second, 1 sorted for a slow cycling, OPN-ve, smdl ce11 population with low granularity and protein content, identified previously in FRCCs (Chapter II; Zohar et ai., 1997) der2 days of culture, a time at which enrichment with osteoprogenitor cells was anticipated (Aubin et al., 1992; McCulloch and Tenenbaum, 1986). Although flow cytometry based on selection of cells for bone heage markers is potentially more discriminating (e-g. Long et al., 1995) and can isolate cells capable of producing bone nodules in vitro (Turksen and Aubin, 1991), nich cells are aiready cornmittecl to osteoblastic differentiation and lose their osteogenic capacity following flow sorting (Twksen and Aubin, 1991). Nevertheless, despite using relatively atraumatic procedures for ce11 separation, ce11 viability was towest in the S subpopulation (60%), which may reflect the paucity of ce11 attachrnent receptors such as CD44 andor the inclusion of small apoptotic cells. Cell Characterizution Although multiple ce11 lineages, including adipocytes, chondrocytes and smooth muscle cells were generated when S cells were grown in culture, 1 focused my studies on the more prominent, osteogenic potential of these cells. When grown on either plastic or collagen substrata, S cells exhibited the sequential expression of phenotypic markers associated with the progressive phases of osteogenesis that have been characterized in UIlfrilctionated calvari al ce11 populations (Lian and Stein, 1992; Owen et al., 1990). Proliferation was the dominant process in the first 48 h of al1 cultured subpopulations. As shown in double labehg experiments, %O% of S cells proliferated but did not exhibit AP activity, which is an early marker of osteogenic differentiation. The major shifk in AP activity and proliferation of S cells over the in culture indicate that some of these cells, which initially did not express OPN or collagen type 1, have the ability to rapidly mature and differentiate, as would be expected for osteoprogenitor cells. Notably, there appeared to be an apparently reciprocal relatiomhip between the relative proportions of proliferating L and S subpopulations of FRCCs cultures. On day 2 the L subpopulation exhibited the highest proportions of proliferating, AP positive cells whereas the S population contained the lowest proportion of these cells. As cultures approached the minerakation stage, the L population, as well as the parental FRCCs population, showed large reductions in the proportions of dual labeled cells while the S population exhibited very high proportions of dual labeled cells (60%). However at the time of bone nodule mineralization, al1 subpopulations exhibited increased proliferation, indicating that bone nodule- forming cells Mght undergo c lonal expansion during bone nodule formation. in support of this concept a quiescent ce11 population has been shown to proliferate extensively at this phase of culture (Zohar et al., 1997) while clonal expansion has been observed at a comparable stage of adipocyte development (Vasseur-Cognet and Lane, 1993)- Whereas OPN, type 1, II and III coilagens and AP activity were initiaüy absent in suspensions of sorted S cells, these proteins were rapidly induced on plaiing- Indeed within 24 h, there was strong staining for types 1 and II collagen and OPN. Thus attachment and spreading at low plating density promoted the differentiation of S-ce11 subpopulation progenitors into various Lineages of which osteogenesis appears prominent. In this context the use of monoclonal antibodies to detect differentiation stage-specific markers has been shown to be a usefid approach for isolating the progeny of stem ceils in hernatopoietic and epithelial tissues. However in bone ce11 lineages, restriction points have been determined only for cells that are aiready cornmitted (Long et al., 1995; Wetterwald et al., 1996). Thus the use of known early expression markers by osteoblast lineage cells (Turkçen and Aubin, 1991) enables isolation of osteoprogenitors but not of stem cells (Potten and Loeffler, 1990). Identification of novel early proteins in this lineage might be related to the unique properties of the matrix-dependent differentiation process in osteogenesis. As shown in this study only S cells (S and L-ve subpopulations) were able to populate type 1 collagen-coated dishes and fonn a significant number of bone nodules. Evidently type 1 collagen substrata (Lynch et al., 1995) drive osteoblast lineage cells to rapid maturation and terminal differentiation which in turn lead to either a lack of bone nodule formation in highly differentiated cells (i-e. the L subpopulation) or a limited number of bone nodules in more heterogenous cells (i-e. the S-ve subpopulation). Diversity of Usteoprogenitors It has been suggested that at low plating densities bone nodules are produced nom cells which arise from a single osteoprogenitor (Bellows and Aubin, 1989). To estimate the relative proportions of osteoprogeniton in the sorted populations, 1 used a Iuniting dilution analysis. Cornparisons of stem cell criteria for S and L populations Sorted 5 Plated S Plated L - - - Cytology Protein ce11 content 4- +i++ Ce11 size + ++++ CytopIasmicgranulari t y + ++++ Nuctearkytoplasm ratio High Low Proliferation S-phase cells 80% 37% Ce11 cycle G&a Ali stages All stages Ail stages Double labeled 3H/M 3% 25% Substratc Plastic dishes ++++ ++ CoUagen-I coated dishes ++++ I Differentiation assays # Bone noduleslfield 20 1.5 Area of hnenoduies/md 11-5 1-61 Substrate: Ekme nodules on col lagen dishes ++++ I Bone nodules on plastic ++++ + Ostmprogenicor kequency 1:202 1 A750

Alkaline Phosphatase œ + (8%) ++ (40% Self-renewal fd passage soning punty 43% 0% # Bane nodules/field 5.5 0.00 Pluripotentiality Bone ++++ + Cartilage +++ +/- Adi pocyc es ++ +

Table m. 2- Characterization of S and L CeU Subpopulations. For each parameter, intensity or level of expression is desmias: - none, + low, t+ medium, * high, * very hi& (%) indicates percentage of cek expressing the specific phenotypic marker. 100 ceils were exarnined in ûiplicate fields in 8 weUs analyzed for each group. Meanrres of proliferation, proportion of S-phase and double- labeled cells are expressed as percentages. Numbers and area of bone nodules reported are per microscopic field, Osteoprogenitor firequencies were computed fiom Limitmg dilution analyses and are expressed as prevaience of progenitors in the whole ceii popdation that are capable of fonning bone nodules. In criteria for self-renewai, sorting purity was caiculated From the proportion of S ceUs tbat exhiiited the same scatter characteristics as the initial sort Collectively, the data show that S-cek are enriched for stem cell rnarkers as desrnid by Potten and Loenler (1990). Data fiom Chapter II and III are combined in the table- The estimated proportion of progenitors in the S cultures (1 :202;or 1: 120 if corrected for cell viability) was three-fold higher than the parental, FRCCs population. However as differentiation of multiple lineages evidently occurs in the S-cell subpopulation, the number of stem cells in the S subpopulation is likely to be much higher than that indicated simply by the proportion of osteogenic progenitors. Previous studies have indicated that osteoprogenitor cells in fetal rat calvarial cultures are alkaline phosphatase positive and have limited self-renewal capacity (Turksen and Aubin, 1991), characteristics which are consistent with the L cells described in rny report. However the S cells also generate bone nodules albeit at a much higher fkequency and unlike L cells, are capable of extensive self-renewal. Further, the nodules produced by the S cells are 10-fold larger than the L cells, indicating that the osteogenic, transit amplifjing cells produced by the S cells undergo at least 3 more ce11 divisions than the progeny of L cells. Thus when nodule formation, nodule size and self-renewal capacity are used as the criteria for osteoprogenitor cells, it is evident that there are different classes of progenitor cells which can ultimately produce bone-forming cells. Further, the wide phenotypic differences between the sorted L- and S-ce11 populations suggest the existence of very different types of subpopulations. Notably, stem cells alter their behaviour markedly when their environment or cornpartment size is altered (Potten and Loeffler, 1990). Consistent with this prediction and as discussed above, 1 observed dramatic phenotypic changes of S-cells within 12h after plating. These alterations were not observed in L cells, indicating that at least in culhue, the progenitors in the L- and S-ce11 populations behave very differently. S?romal Progenitor Cells Collectively, these studies have charactenzed a relatively undifferentiated ce11 population derived £iom fetal rat periosteum. The features of small size, low granularity, low cytoplasmic to nuclear ratio and undetectable expression of osteopontin, collagens and alkaline phosphatase activity are consistent with the expectations of a mesenchymal stem ce11 (Table III. 2). Further, on plating, these cells proliferated rapidly and generated new self-renewing cells as well as transit amplifjmg cells (lineage directed ceils) that expressed early lineage markers, such as collagen type 1, AP and OPN for the osteogenic lineage. Further differentiation of thae cells was promoted by the formation of a coilagen matrix beneath the cells, consistent with the observation that tissue nodules are formed following the production of an extracellular matrix by fully-differentiated cells (Aronow et al., 1990). At this stage it is conceivable that the size of the tissue nodule is also increased through the clonal expansion of osteoblast precursors. During the differentiation process, a large number of cells at different stages of differentiation are produced: the more highly di Eerentiated cells (i .e. the L population) having a more limited pro liferative capacity. If this paradigm of cellular differentiation in vitro parallels the normal processes occumhg during the maintenance and repair of stromal tissues in vivo, then it might be expected that the heterogeneity of ceIl populations isolated ftom stromal tissues (Liu et al., 1994) is a consequence of progressive differentiation and one in which the number of stem ceUs is retained at low levels. The application of the flow cytometry sorthg method described here for the enrichment of stem cells provides a simple, reproducible approach to help delineate the stages of bone ce11 differentiation. Further, the ability of flow cytornetry to enrich for stem cells has significant potential for enhancing clinical procedures aimed at regenerating stromal tissues, particularly in the elderly where the numbers of stem cells are believed to be low. CHAPTER IV

Flow Cytometric Analysis of rhOP-1 (BMP-7) Responsive Subpopulations from Fetal Rat Calvaria Based on IntracelMar Osteopontin Content ABSTRACT The Bone Morphogenetic Proteins (BMPs) are characterized by their ability to induce both chondrogenic and osteogenic differentiaîion of mesenchymal ceUs in vivo and in +o. Primary cultures of fetal rat calvarial ceils contain a broad spectrum of osteogenic ce& at various stages of differentiation but the cesponsive subpopuiations are incompletely characterized. To identify responsive ceils in osteogenic ceil differentiation 1 treated fdrat calvarial (FRCCs) ceils with recombinant osteogenic protein-l(rh0P-1; BMPs) and used flow cytometric analyses of mtraceilular OPN, and cartilage and bone nodule formaton to evaluate the effécts. When administered as a singie dose at confluence, MP-1 stirnulated bone nodule formation in fetal rat calvarial cultures dosedependently. To determine the response of osteogenic subpopulations at two discrete stages of differentiation, fetd rat calvaria ceils were cultured for 2 days (prolifdve stage) or 12 days (early mineralization stage) and treated with 100 @ml rhOP-1 for 12 h prior to analysis by flow cytomeûy. Flow cytometry analyses of cell suspensions revealed that &OP-1 increa~edthe total protein content of cells, selectively increased the mean expression of OPN and the size and granularity of OPN expressing cells, particulady at day 12, consistent with a stimulation of osteogenic differentiation and matrix formation. Pulse administration of 100 ng/ml &OP-1 to sorted, OPN-ve subpopulations enricheci for stem cek reduced by more than Cfold the number and size of bone nodules while promoting chondrogenesis and adipogenesis. In con- a pulse administration of rhOP-l to more Merentiated, large OPN+ve cells increased bone nodule formation two-fold. Continuous administration of 100 ng/ml rhOP-1 to the large osteopontin positive and srnd OPN-ve ceil populations obliterated bone nodule formation and pmmoted chondrogenesis. Thus, pulse administration of &OP-l promotes osteogenic differentiation of cells committed to the osteogenic lineage whereas undifferentiated periosteal cells are induced to dinedate dong the chondrogenic pathway. In con- continuous exposure to hop-1 promotes chondrogenesis in populations of conmitteci osteogenic cells and undifferentiated periosteal cells. INTRODUCTION The BMPs are a sub-group of the transforming growth factor+ (TGF-P) superfamily of cytokines that mediate epithelial-mesenchyrnal interactions duruig growth and development (Womey, 1992; Reddi and Cunningham, 1993). Although BMPs have a broad range of activities in developrnent they are characterized by their ability to induce bone formation (Reddi, 1994). Recombinant human rhOP-1 @OP-1; human BMP-7) has potent bone inductive activity both in vivo and in vin0 (Sampath et al., 1992). In FRCCs and nematal rat calvarial celi cultures, rhOP-1 increases ceii proliferation, stimulates expression of osteogenic markers, such as aikaline phosphatase, osteocalch and bone sialoprotein and, in the presence of organic phosphate and ascorbate, rhOP-1 increases the number and size of mineralized bone nodules and their rate of formation (Sampath et al., 1992; Li et al., 1996). When implanted at ectopic sites BMPs promote endochondral bone formation by stimulahg undifferentiated mesenchymal cells to differentiate and produce cartilage, which is subsequentiy replaced by bone (Reddi, 1981). However, when introduced directly into bony sites BMPs stimulate the formation of bone without initial cartilage formation (Cook et al., 1995). Studies in vitro have shown that BMPs can promote cellular differentiation dong multiple pathways depending upon the characteristics of the responding ce11 population. Thus while BMPs generally stimulate bone formation in cultures of osteogenic cells (Sampath et al., 1992; Knutsen et al., 1993), rhOP-1 can also promote chondrogenic differentiation (Asahina et al., 1993). These data support the concept that osteogenic precursor cells are heterogenous (Aubin et al., 1992) and that rhOP-1 increases bone formation through its action on a subpopulation of osteoprogenitor ceils (Sampath et al., 1992). However, the specific target ceil populations in osteogenic tissues that are regulated by BMPs have not been characterized. The complexity of osteodifférentiation (Lian and Stein, 1992) and the broad distriiution of maturational stages of osteogenic ce11 populations (Aubin et al., 1992) are central problems in studies of cytokine-responsiveness of cells. To circumvent some of these ditncdties 1 have used fîow cytometry and the expression of intracellular osteopontin (OPN) and osteonectin (ON) to study osteogenic differentiation in discrete subpopdations of FRCCs (Zohar et al., 1997a). Atthough OPN expression is not restricted to bone it can provide a usefül marker for the early differentiation of osteogenic ceh (Mark et al., 1985; Yoon et al., 1987; Yao et al., 1994). In previous studies I have isolated a population of small ceils that do not express OPN and that are enriched with stromai stem celis (S celis) capable of generating bone, fat and cartilage (Zohar et al., 1997b). 1 have now used a combination of flow cytometry and expression of OPN to deheate BMP-responsive cells in osteogenic cultures and to determine the differential responses of flow-sorted subpopulations to BMPs. In combination with colony assays for bone and cartilage formation 1 showed that a single administration of rhOP-l stimulates chondrogenic differentiation in startùig populations of cells that are srnall in size, have low abundance of cytoplasmic organelles, do not express OPN and have characteristics of stem cells. In con- osteogenesis is stimulateci by the expansion of cells that express OPN in populations committed to the osteogenic lineage. Continuous rhOP-1 treatment on the other hand promotes chondrogenesis in both undifferentiated S cells and in committed osteogenic populations. MATEIUALS AND METHODS Cell cuitures Fetal rat calvarial cell populations were prepared by five, sequential enymatic digestions (I-V) of cdvariae hm 21-day-old fehises of tUmed-pregnant Wistar rats, as descriied in detail previously (BeUows et ai., 1986; 1990). Care was taken during dissection of the calvaria to ensure that cartilaginous edges were discarded hm the tissues. Cell populations fiom digestions II-V were pooled, plated in T-75 flasks and grown in a- minimal essential medium (a-MEM)containing 15% inacîivated fetal bovine serum (FBS) and antibiotics (100 rng/ml penicillin G, 50 mghl gentamycin sulphate, and 0.3 rng/ml fungizone). Cells were grown at 3TC in a humidifieci atmosphere of 95% air/5% CO, and media were changed every 2 or 3 days. Mer 24 h incubation, attached cells were washed with PBS to remove non-viable cells and then released with 0.01% ûypsin in citrate buffer. Aiiquots were electronically comted (ZM Coulter Counter) and replated in T-25 flasks at a density of 7.5x104 cells per fiask. Four independent ce11 preparations were used in these studies. Culture conditions were identical in al1 experirnents except where detailed below. Cells were grown continuously for periods of 2 and 12 days for single ce11 analyses and for preparative flow cytometry. Cells were grown for 16 days for studies of bone nodule formation. The proliferation and differentiation responses of FRCCs to rhOP-1 were also compared with MC3T3-E 1 cells, a well-characterized osteogenic ce11 line hmembryonic mouse calvaria (Sudo et al., 1983), to determine the most appropriate osteogenic ce11 systern for study. rh OP-l Treatment Recombinant human rhOP- 1 (rhOP- 1; BMP-7; fkom Creative Biomolecules, Hopkinton, MA) was stored at -20°C in 50% acetonit.de containing 0.1% trifluoroacetic acid. For studies of bone nodule formation, 0, 10, 25, 100 or 250 @ml rhOP-1 was added as a single administration when cells were plated or at confluence (pulse). For flow cytometric analyses, 100 ndml of rhOP-1 was added in Eesh medium 12 h before harvesting cells that had been cultured for 2 or 12 days. This concentration of rhOP-1, and the timing of administration, was optUnized in earlier experiments (Knutsen et al., 1993; Sodek et al., 1994). Contml flasks received the solvent vehicle ody. Analysis of lnhricelltil~rand Secreted OPN The intracellular content of OPN was compared to secreted OPN in subconfiuent day 4 FRCCs cul- in order to ver@ the authenticity of the OPN antibody staining and the validity of imrnunostaining for intracelldar OPN. Cells in 60-mm dishes were washed twice with a-MEM containing 5% FBS. Conditioned medium was coliected after 3 h and the cells were washed three times with cold PBS and then collected by scraping in 1 ml of 50 mM Tris-HC1 10 mM CaCl,, pH 8. The cell layer hction was sonicated twice for 10 sec (Branson Sonifier seîting #7) and the supematant clarified by centrifugation at 10,000 x g for 7 min on a microfùge. Notably, sonication was found to solubilize the cellular OPN more efficiently than detergent extractions. Ceil lysates and medium samples were analyzed by irnmunoblotting as describeci previously (Zohar et al., 1997a) using an OPN monoclonal anti'body (see below). hunoblotting of medium alone (Le. no cells) containing fetal bovine semm showed no band on the blots, indicating that the immunoblot of ce11 lysates was not detecting contamination hm bovine OPN in the medium. Sarnples of the ce11 lysate and media proteins were also digested with 5 units of thrombin (Sigma) in 10 ml of 50 rnM Tns-HCI, 10 mM CaCI,, pH 8, prior to immunoblot analysis to confirm the identity of the OPN bands. Alknline Phosphatase Expression and Cell Proliferation The effect of rhOP-1 on alkaline phospatase (AP) activity and proliferation (labehg index) in cultures was examined in FRCCs and MC3T3-El ceils, a mouse-derived osteogenic ce11 line. Cells were seeded into 8 well chamber slides (Nunc, Roskilde, Denmark) at 1x1 O4 cells per well. rhOP- 1 at 100 @ml was added to four replicate wells for each experimental group, 24 h before analysis. The fiaction of proliferating cells was assessed by incubating cells with 'H-thymidine (1 pCi/rnl; S.A=20 Ci/mmol) for 12 h before terminating the cultures. Single ce11 emulsion radioautography was used instead of acid precipitation of whole culture labeled DNA to assess more accurately the hction of prolifiig cells in rat osteogenic cultures which are known to contain an abundance of non-proIiferatiag ce& (Zohar et al., 1997a). Slides were nXed in cold 3% padonnaldehyde, pH 7.4, for 10 min and ceiis were stained for AP activity with a naphth01 TS phosphate substrate (0.2 mom) containing Fast Blue BB salts (Sigma) in Tris buffer, pH 9.0. The slides were subsequently prepared for radioautography with liquid exnulsion (NTB-2; Kodak, Rochester, NY), as described (McCulloch and Tenenbaum, 1986). The mean percentage of labeled ceus per microscopie field area (40x objective; triplicate fields for each culture) were determined. Coiorry Assays FRCCs were plated in triplicate 35-mm dishes at 3x10' cells per dish and the cultures augmented with 50 rn@ sodium ascorbate and 10 mM sodium P- glycerophosphate (Sigma) at confluence. &OP-l was added at 0-250 ng/ml either continuously fiom day 1 or as a 244 pulse at confluence. The cultures were temzinated on day 16 (the earliest time when nodules had fomed at ail concentrations), fixed ovemight in neutral buffered formalin, and stained with Von Kossa's ragent. The number and area of bone nodules were measured in each 35 mm dish using an image analyzer (Bioquant; R & M Biornetrics, Nashville, TN). The nodule area was expresseci as mm2of mineralized tissue per 50 mm' surface area in each 35 mm dish. The mean nurnber * the standard mors of the mean of mineralized tissue nodules and their area were computed. By means of simila. analytical methods, the number and area of cartilage nodules (stained with alcian blue) and of fat-containing cells (stained with Oil-Red) was determined. Flow Cytornetry Cells were harvested with a proprietory enzyme-fiee ce11 dissociation buffer prepared in Ca2'-MC-kee PBS (Life Technologies, Burlington, ON, Canada), washed in the flask for 30 sec to rernove debris and dead ceils, and incubateci with the buffer for 3 to 4 min. To ensure that single cell suspensions were obtained, the cells were transferred to a tube with gentie pipetting. Celis were fked with an quai votume of 2% paraformaldehyde in ca2+-~g-hePBS for 30 min and permeabilized with 0.01% Triton-X to facilitate entry of antridies and thereby measure total cell OPN and ON. hunocytochemical analyses of cells consistecl of two steps which uicluded staining for bone rnatrur proteins and staining with a DNA specinc dye for ceil cycle analysis. Different cell samples were used for stainuig with the two antiodies to eliminate problems with crossover emission hmthe FITC and PE channels, detennined previously in double labeling experiments (aha.et al., 1997a). The two anfibodies used in this study were a mouse anti-rat OPN monoclonal anhiody (MPI5101; obtained brn the Developrnental Studies Hybridorna Bank, Johns Hopkins University, Baltimore, MD under contract hmNICHD; dilution of 1:$O0 in 0.25% w/v BSA in Ca2+-M$-fke PBS) and an affInity-purifïed polyclonal rabbit anti-porcine SPARC/osteonectin (ON) antibody (Domenicucci et al., 1988), diluted 1:20 in the same 0.25% BSA solution. mer fixation, cells were washed with a 0.25% BSA solution, centnfùged at 200 g and the pellet incubated with 2 ml of either OPN antibody for 30 min or SPARC antibody for 1 h at 4"C, foilowed by a 10-min incubation at room temperature (2I0C).The cells were washed with the BSA solution and re-pelleted. The ceils incubated with OPN antibodies were stained with FITC- conjugated sheep anti-mouse antibodies diluted 1:100 in the BSA solution for 30 min at 4OC. The cells incubated with ON antibodies were stained with R-phycoerythrin-conjugated (PE) goat anti-rabbit F(ab), fragments diluted 1:20 in the same BSA solution for lh at 4OC. Cell suspensions that were stained for OPN or for ON did not show detectable fluorescence unless the cells were fked and permeabilized, indicating that the proteins as detected by flow cytometry were indeed intracellular as has been shown in detail earlier (Zohar et al., L997a). Shed cells were washed with Ca2+-~g-fieePBS, re-pelleted, re-suspendeci in 0.7 ml of 4, 6-Diamidino-2-phenylindole dihydrochloride (DAPI; Boehringer Mannheim, Germany; 1 mghi £inal concentration) and incubated in 0.2% Triton X-100 for 10 min at room temperature before fiow cytometry. One sample of ceils was assessed for total protein content. These cells were washed immediately after fixation in 2% pacafonnaldehyde with Ca2'-~$-f?ee PBS and re- suspended in a mixture of DAPI and sulforhodamine 101 (Texas red; Molecular Probes, Inc, USA; 20 mg/d final concentration). Ceils were incubated for IO min at room 94 temperature before flow cytometry anaiysis. Flow cytomeûy was peforrned on a FACstar Plus flow cytometer (Beçton Dickinson Tmmunoctochemistry Systems, Mountainview, CA) equipped with two argon lasers. Two color excitation was used for analyzing the celis: the 488-nm beam was wed for excitation of FITC-conjugated and PE-conjugated second antiibodies and for sulforhodamke. The 362-nm beam IUV) was used for DAPI. FITC fluorescence was measured at an emission range of 510-530 nm with a band pass fiIter in the emission path and PE fluorescence at 550600 m. Thresholds were set for each analysis using the same cells fixed and stained with FITC or PEtonjugated antibodies alone. DAPI staining was verified before each analysis by analyshg caK thymocyte nuclei as standards. Analysis windows above pre-determinecl background thresholds were established for the following parameters: FITC fluorescence; PE fluorescence; sulforhodamine fluorescence; forward light scatter (FSC) and side scatter (SSC). Analyses were restncted to cells that exhibited diploid (2N; G, and Gotells). tetraploid (4N; G*, cells) or intermediate DAPI staining (S- phase cells). Light scattering was computed for cells above the detection threshold for FITC or PE fluorescence. A second group was assessed for cells below the threshold (Le. negative staùiing cells). Cell Sorting Attached cells were harvested using 5 mi of 0.01% trypsin in citrate bufKer and cells fiom 5 T-75 flasks (4x106 cells) were re-suspended in 2 ml a-MEM (phen01 red-free, to eliminate artefactual fluorescence during flow cytometry) containing 15% filtered FBS and 10% antibiotics. Sorthg was performed on a FACStar Plus flow cytometer (Becton Dickinson) as described above. Gaihg windows were established for fomd light scatter (FSC) and side scatter (SSC). Particles with an average sue <5mm (determined by running standard size beads) were excluded. Two major subpopulations were sorted: cells with the lowest 15% FSC and lowest 15% SSC in the population (S cells) and the cells with the highest 15% FSC and highest 15% SSC (L cells). AU cells were collected in glas tubes containing 3 ml a-MEMwith 15% FBS and 10Y0 antibiotics. For continuous data, means and standard arors were cornputecl. Cornparisons of two groups were analyzed by unpaired t-tests and for more than one group analysis of variance was performed. At least 3 separate experiments were conducted for each assay.

RESULTS Assasrnent of the FRCCs Model To confimi that fetal rat calvarial cells (FRCCs) were an appropriate systern for these studies 1 first examined the growth and differentiation of bhly isolated FRCCs with MC3T3-El cells in response to rhOP-1 using 'H-thymidine emulsion radioautography as a measure of cell proliferation, and allcaline phosphatase activity as a. indicator of osteodifferentiation. Following a 12 h 3H-ttiyrnidine labeling regimen the labeling indices (LI = No. labeled ceildNo. total cells) of the cultures were determineci; ceUs with four or more silver grains per nucleus were defineci as being labeled (Fig IV. 10).For FRCC, the highest LIS were observed during the fbt 2 days of culture (0.6). The LIS decreased significantly after 3 days (pc0.01) and reached very low levels by day 6 (~0.15).rhOP-1 significantly reduced the LI on day 3 and day 4 (pcO.02). For MC3T3-E1 celk 1 observed a very sirnilar reduction of proliferation over increased the of culture (Fig IV. 1B) but the MC3T3-El cultures exhibited larger variances and the rhûP-1-induced increases of labeling indices were not statistically signifïcant. In FRCCs there were low percentages of alkaline phosphatase positive cells (Fig IV. 1C) on the ktday of culture but the nurnber of positive cells increased >IO-fold on the fourth day and reached maximal levels on day 5 at which time almost al1 cells were AP+ve. The percentage of AP+ve cells in FRCCs cultures was increased significantly by rhOP- 1 korn days 14Q~0.01). A sirnilar pattern was observed with the MC3T3-El cells but as with the proliferation data the variances were large and statistically significant differences between controls and rhOP-1 treated cultures were not observed until day 7 (Fig TV. ID). Consequently, in view of the large variances and the lack of statistically significant differences afkr rhOP-1 treatment in MC3T3-El cells, as well as the difficulty in 96 perfomiing assays of minefalized tissue formation, 1 perfomed ail subsequent analyses on FRCCs. Mineralized Tissue Formation To evaluate dosedependent effects of &OP-1 (0-250nghnl) on osteogenesis in FRCCs, bone-like nodules were stauied with von Kossa's reagent and anaiyzed after 16 days in culture. At this tirne nodules had fodin ail groups. albeit at very low numbers in conml cuitures (Fig IV. 2). Measuring nodule formation at this theminimized errors due to coalescence of nodules, which increases the apparent size of nodules while reducing their apparent number. A dosedependait increase in the number of mineralized nodules was observed when the &OP-1 was administered as a pulse at confluence (Fig W.2), although the increase in the number of nodules between 10 and 250 ng/d rhOP-1 was relatively small (50% increase; pc0.05). Compared to controls, the average size of nodules was increased 15-fold by 10 ngM rhOP-1, but no fùrther increase was observed until the dose reached 250 nglml rhOP- 1. OPN and ON Content Total ce11 protein content was fht determined by sulphorhodamine staining to normalize immtmofluorescence for OPN and ON. In control and &OP-1 treated cells, protein content was low at the proliferative stage of cultures (day 2) but was nearly doubled when rnatrix production was underway (day 12). Cell cycle analysis showed progressive increases of protein content during transit from G, to G2and through mitosis, as has been shown earlier for fibroblastic cells (Darzynkiewicz et al., 1982). The rhOP-1 treatment increased protein content by 10- 15%. Since previous studies had show that OPN mRNA expression in confluent FRCCs was increased 6-12 h after rhOP-1 administration (Li et al., 1996), cells were treated with 100 ng/d rhOP-l for 12 h before flow cytometric analysis. More than 75% of cells at both 2 and 12 days of culture were positively stained for OPN (i.e. OPN+ve; Fig TV. 3A). rhOP-1 increased the percentage of OPN+ve cells modestly (by -5% at day 2 and -8% at day 12; pX.2). In contrast to OPN, anaiysis of another bone matrix protein, SPARC/osteonectin (ON), showed no positively stained cells in the control group and less than 5% positively stained ceUs at day 2 in the &OP-1-treated group. There 97 were higher proportions of ON+ve ceiis at &y 12 (1420%) compared to &y 2 but rhOP-l did not signincantly increase the % of ON+ve cells 0.2). To detennine the relative amounts of OPN and ON immunolabeled cells were also analyzed for mean fluorescence inteLlSity (Fig W.3B). As observed previously (Zohar et al., 1997a), the mean OPN fluorescence per ce11 for celis stained above threshoId values was not related to the proportion of cells in the whole population that were positively stained for OPN. rhOP-1 increased OPN content on both day 2 and 12 @<0.01) but the effect was more pronounced when cultures approached the mineraiization stage (60% increase at day 12 compared to 35% at day 2). At day 2, the very low percentage of cells positively stained for ON generated fluorescence signals only marginally above threshold values and again at day 12, no significant changes in ON content were apparent after treatment with rhOP-l (Fig IV. 3B). Cell cycle analyses showed that at day 2 and 12 the mean OPN expression increased with transit hm G, to mitosis in both control and rhOP-1 treated cens. This increase occurred at day 2 and 12 and the inductive effects of rhOP-1 on OPN content were evenly distributeci thmugh the different phases of the cell cycle. Characteri,-aion of lntracellular and Ectracelluhr OPN To characterize the intracellular immunoreactive matenal detected with the OPN monoclonal antibody used in the flow cytometry studies, ce11 lysates and medium samples were analyzed using Western blots (Fig N.4). Al1 samples exhibited two forms of OPN corresponding in size to the low phosphorylated OPN (LP-OPN), which was the major isoform in the FRCCs cultures, and to the hi& phosphorylated OPN (HP-OPN), which was the minor isoform (Wrana et al., 1991). The identity of the immunoreactive bands from both cellular and medium as OPN was cobed by their susceptibility to thrombin cleavage (Fig IV. 4). As the imrnunoblot data of secreted OPN showed that the antibody used for flow cytometric analysis of inûacellular OPN recognized a simila. molecular mass isoform as the secreted OPN,and both secreted and intrace1ldar OPN exhibited the same thrombin sensitivity, 1 was coddent that the flow cytometric measurements of OPN reflected the levels of an authentic intraceUular fom of OPN. Morphorogical Feaiures of FRCCs Forward light scatter (FSC), used as a masure of relative ceil size and side scatter (SSC) as a measure of cytoplasmic granularhy, reflect the complexity and relative abundance of cytoplasmic organeiles inciuding rough endoplasmic reticdum, lysosomes and Golgi apparatus. Consistent with these observations, day 2 celis stained for OPN (OPN+ve), a Merentiation marker for osteogenic cells (Yoon et al., 1987; Lia. and Stein, 1992; Yao et al., 1994), were more than 2-fold larger and more granular than OPN-ve cells on day 2 @<0.001; Fig N. 5). Treatment with rhOP-l slightly reduced the FSC (by 7%) and SSC (by 16%) for OPN+ve cells (p<0.05) but had no effect on OPN-ve ceils at day 2. in contrast, rhOP-1 increased FSC (by 60%) and SSC (by 50%) for OPN+ve cells at day 12 @<0.001). The effect of &OP-1 on OPN-ve ceils was smaller than the OPN+ve cells (35% increase for FSC and 28% for SSC; both ~~0.01). As expected for proliferating eukaryotic cells, cell cycle andysis showed that ce11 size gradually increased hmG, through to mitosis in the presence and absence of rhOP-1. OPN-ve ceHs were restricted to the G, and S phases in day 2 cultures and were much smaller than OPN+ve cells at each cell cycle stage. Treatment with rhOP-1 did not rnarkedly afEéct the size of cells in day 2 cultures. In con- rhOP-1 strongly increased the size of cells at day 12 and this effect was found for al1 phases of the ce11 cycle. Cells expressing SPARC/osteonectin did not exhibit any changes in FSC or SSC and there were no ciifferences in the size or granuiarity of ON+ve and ON-ve cells. Further, FSC and SSC meastuements of ON+ve cells did not show any relation to the whole population size measurements or mean ON expression, as observed with OPN+ve cells, indicating the selectivity of the effects of rhOP-1 on OPN expression and demonstrating the sensitivity of the flow cytometric analysis. Responses of S and L Cells to rhOP-l 1 have shown previously that OPN-ve cells nom FRCCs comprise a subpopulation e~chedfor cells with stem cell-like characteristics (S-cels; Zohar et al., 1997b). These cells can be separated by flow cytometry from large OPN+ve cells (L cells) which have the characteristics of more di fferentiated cells. 99 Fig IV. 1- Ceii ProMeration and AIkaline Phosphatase Expression in FRCCs and MC3T3-E 1 Cells. A,B- Ceii proliferation (measured as mean labelhg indefi s.e.m) in response to rhOP-1 at ciiffernt stages of ceiiuiar differentiation in cultures of FRCCs cek was determined fouowing a 12 h incubation with [3w-thy"dine. C,D- The percentage of ce& expressing alkaline phosphatase, a marker of bone cell differentiation, was detemiiaed at the same time points analyzed for ceiiuiar prolifération using a Napthol AS phosphate subsîrate containing Fast Blue BB salts (mean % AP+ve ceIls). INodule No-- Pulse a Nodule No.-Contlnuous Nodule Are* Pulse Nodule Area- Contlnuous

O 10 25 100 250 OP-1 Concentration (ng/ml)

Fig IV. 2-Bone Nodule Formation induced by rhOP-1 in FRCCs Cultures. The number and area (m')of bone nodules formed after &OP-1 administtation as a single pulse, given at confluence was determineci der 16 &YS of cuiture. Bone nodules were visualized by staiuing with Von Kossa's reagent and measured by automated image anaiysis. nie number of bone nodules were counted in triplicate fields in three dishes for each group. 12 Day

Fig IV. 3- Flow Cytometric Analysis of the Relationship between Celi Nudm and intracellular OPN in FRCCs. Foiiowiag immunolabeling of intraceiiular osteopontin (OPN) and SPARC/osteonectin (ON), single ceii suspensions of fetai rat calvarial cek, cultured for 2 and 12 days, were dyzed by flow cytometry in four independent experiments. FIuorescence intensity si@ above the threshold Ievei, deterrnined fiom the fluorescence obtained when using the second antiiy alone, were used to identify cells staiuing positively for OPN or ON- A- Histogram showing the proportion (%) of celis in the whoIe population that stained for OPN or ON before and dertreatment with 100 og/d rhOP- 1. EL Histogram showing the effects of rhOP-1 treatment on the arnounts of intraceiiular OPN and ON, as indicated by changes m fluorescence intensity. S ceils hm 2 &y cell popdations represent the smallest 15% of the whole FRCCs population while L cells represent the largest 15% of the whole ceii population. When S and L cell subpopulations were isolated hmday 2 cultures of FRCCs the S ceb showed a large capacity for generating bone-like nodules (Fig IV. 6,7), with a smaiier capacity for generating cartiiage and adipocyte nodules. In contrast, L cells generated Iow nmbers of smWbone nodules (Fig TV. 7). When administered as a single pulse at plating the rhûP-l almost completely blocked bone nodule formation in S cells with an associateci 50% reduction Ui the average size of the colonies w0.05). Similarly, the number of adipocyte colonies was reduced by 40% and the size was reduced by >3-fold in S cells w0.05). However, this treatment increased the nimiber of cartilage nodules >cl-fold @<0.01) and increased their average size ahost 2-fold. In contras&pulse administration of rhûP-1 to L cells increased bone nodule number almost 2-fold @

mm-' i

Fig W.4-Western Blot Analysis of OPN in FRCCs Exîracts and Conditioned Medium. Confluent FRCCs were incubated in 5% senun for 3 h to obtain conditioned medium and the ceU layes were washed Ceil extracts and conditioned medium ~a~npleswere analyzed for OPN by immunobloaing before and after digestion with thrombin (Th). Two irmnunoreactive bands corresponding to low and hi&-phosphorylated forms of OPN were observecl; the slow migrating OPN was more prominent Both bands were susceptible to thrombin digestion, Fig IV. 5-Relationshp between inûaceiiuIar OPN and Size and Granuiarity of FRCCs. Flow cytometry of FRCCs cultured for 2 days was used to analyze (A) forward light scatter (a measme of ceii size/voIume changes) and (B) side scatter (a measure of cytoplasmic granuiarity) of OPN+ve and OPN-ve cek. Redts are expressed as the mm.e.m. J3g W.6- Phasecontriut Micrographs of Bone and Cartilage Colonies formed by S and L Cek. A-D-Cultures shedfor mineralized tissue formation with von Kossa's reagent. EH-Cultures stained with aician blue for adagecolonies. A- Untreated S ceU cultures generated a high number of iarge bone nodules. Lower numbers of smaiier bone noduies were observed when cultures were pulse-treated with IO0 ng/ml rhOP-1 (Ba),while only background staining with Von Kossa's reagent was seen in cultures given rhOP- 1 conhuously (C). Cartilage noduies in weated S ceU culûms (E)and cultures pulse-treated with Blue colonies. rhOP-1. . at coduence (F) generated smali numbers of Aician stakùng Continuous achmstration of rtiOP-1 to S ceii cultures (G) produced the largest cartiIage colonies, while ceLis puIse treated with rhOP- 1 at pbting (H) extilïited large numbers of small cartilage colonies. DISCUSSION A principal characteristic of the BMPs is their ability to promote bone formation (Reddi, 1994) but the wide distribution of maturational stages of ceU populations in osteogenic tissues has precluded a detailed description of the target populations of these cytokines. Previous studies of rhOP-l modulation of osteogenic differentiation have analysed ce11 lines (Asahba et aï., 1996) or whole populations of neonatal rat calvarial cultures (Asahina et al., 1993). Similar to these earlier studies 1 found that the effects of rhOP-1 on bone formation are dependent upon the developmental stage of the culture. The novelty of the data presented here is in the use of flow cytometry to study rhOP-1 regulation of an intracellular differentiation marker, OPN, in a whole penosteal ce11 population and then to assess the effects of rhOP-1 on sorted subpopulations. Although flow cytometry has considerable analytical discriminative power, based on an ability to study simultaneously, the size, cytoplasmic complexity and phenotype, the technique has seen limited use in studies of osteogenesis (Zohar et al., 1997a; 19971). OPN Content Cognizant of the bimodal expression of OPN during osteogenic differentiation (Lian and Stein, 1992; Yao et al., 1994), 1 analyzed OPN expression at the early proliferation stage (Le. 2 days) and at the early stages of bone nodule formation (12 days) in whole FFKCs populations. At both 2 and 12 days after plating, the responsa to rhOP-1, indicated by alterations in intracelluiar OPN content were selective when compared with ON. Further, quantitative differences in OPN expression were evident at days 2 and 12 which appear to reflect rhOP-1 effects on osteogenic differentiation and matruc formation. At day 2, the mal1 increase in the proportion of cells expressing OPN and in OPN content, without significant changes in ce11 size and granularity, suggest that only a small proportion of cells respond within 12 h to rhOP-1, as measured by OPN content. Similar effects on OPN were observed with dexamethasone, which is known to stimulate osteogenesis in these cultures (Bellows et al., 1990). Thus, 1 suggest that these rhOP-1 responsive cells are likely to comprise committed osteogenic ce11 populations. in contra$ on day 12, in addition to the marked increase in OPN expression, there was a large increase in the size and granularity of 1O7 Bone Nodules

-- Cartilage Nodules

te Nodules

Control Pulse Puise Contin. (piating) (cod.)

Fig IV. 7- Analysis of Colony Formation by S and L Ceiis in Response to rhOP- 1. The formation of bone, cartilage and fat tissue nodules formed by the S and L populations isolated from day 2 cultures of FRCCs was detemimed foiiowing staining with Von Kossa's reagent ,Alcian Blue and Oii Red , respectively. The effects of &OP-1 (100 n@), administered either as a pulse at plating, or as a continuous admmistration, or a pulse at confluence on the colony n& and area (mm? were determined (mean s.e.xn) by automated image andysis. The nurnber of colonies counted in three dishes for each group in tripiicate fields. OPN+ve cells, similar to results (not show) obtained with TGF-BI, a cytokine which stimulates matrix protein production (Wrana et al., 1991). The response of &y 12 ceils is, therefore, consistent with a response of the more abundant and more differentiated osteogenic cells that are present at day 12 (Zohar, 1997b). Momver, the increase in the proportion of OPN+ve cek is consistent with the increase in both the number and size of bone nodules formed when confluent FRCCs are given a single administraîion of hOP-1 (Fig W.2) and with the proposeci clonal expansion of osteogenic cells shown in a previous study (Li et al., 1996). Cell Sorring The responses to rhOP-1 that were observed by analytical flow cytometry of the whole FRCCs population prompted us to study the effects of rhOP-l on isolated, discrete subpopulations (S and L) sorted by size and grandarity nom day 2 FRCCs populations. The S cells comprise a population of srnall, OPN-ve cells with low granularity and the absence of expression of bone specific proteins that have the characteristics of undifferentiated cells includuig stem cells (Zohar et al., 1997b). Typically, the S cells produce high numbers of large bone nodules in colony assays, consistent with their high capacity for proliferation and differentiation. In contrast, the large OPN+ve cells with high granularity (L cells) sorted fiom the same FRCCs preparations have characteristics of more differentiated, committed osteogenic cells. While the Lsell population can generate bone, the nodules are few in number and srnall, reflecting their more limited proliferative and differentiation potential. The effkct of rhOP-1 on the undifferentiated S cells, as indicated from colony assays, was to promote chondrogenesis while suppressing osteogenesis. Thus, the response of the S cells parallels the effects of rhOP-1 on undifferentiated mesenchymal cells in vivo (Sampath et al., 1992) where the cytokine induces cartilage formation at ectopic sites. However, the suppression of osteogenesis that accompanies the increased chondrogenesis is less dramatic when S cells are given rhOP-l at confluence, presumably when the differentiation of the S cells had progressed. In contrast, rhOP-1 given as a pulse to L cells at plating or at confiuence stimulated bone formation, consistent with the increased bone formation observed with confluent FRCCs cultures in which S cells 109 comprise only a smd proportion of the total population. Notably, continuous administration of rhOP-1 abolished osteogenesis in S and L subpopulations while chondrogenesis was stimulateci, indicating that following an initiai promotion of osteogenic differentiation, the continual presence of &OP-l can suppress bone formation. rhOP-1 Responsive PopuIations A marked diffkrmce in the response of the whole FRCCs ceil population to rhOP-1 and the responses of the S and L subpopulations was observed. Even at low concentrations (10 ng/ml) of rhOP-1, the whole FRCCs ceii population exhibited a several-fold increase of nodule area and nodule number while for the S and L subpopulations these measaues were either increased two-fold (Z. ceils) or were strongly inhibiteci (S cells). Thus, sorting out the mid-size cells f?om the whole FRCCs cell population by flow cytometry reduces the heterogeneity of the osteogenic cell populations and deletes important MP-l responsive cells that are responsible for bone nodule formation. These non-S, non-L cells cm apparently respond to rhOP-1 with a large increase of bone nodule formation but not necessarily with an intermediate step of cartilage formation. The differential responses of specific osteogenic subpopulations is notable because in vivo, when piaced at ectopic sites in an appropriate vehicle, BMPs induce mesenchymal cells to form cartilage which is subsequently replaced by bone, following the developmental pathway of endochodral bone formation (Reddi, 1981). Thse observations indicate that BMPs target undifferentiated stroma1 cells, promothg their differentiation dong a chondrogenic pathway as indicated by the in vzlro studies of Asahina et al. (1993). While previous studies using ce11 lines have aiso shown that rhOP-1 can induce osteoblastic, chondrogenic and adipogenic differentiation that is dependent upon the nature of the target cells (Asahina et al., 1996), here I have specifically identified a subpopulation of undifferentiated normal periosteal cells as the target of rhOP- 1-induced chondrogenesis. Further, 1 have shown that rhOP-1 will promote osteogenic differentiation in cells committed to the osteogenic lineage, consistent with periosteal bone formation in the absence of cartilage when rhOP-1 is administered to bony sites in vivo (Cook et al., 1995). However, these studies also indicate that continuous exposure of osteogenic ceils to BMPs 110 may suppress bone forniaton in committed osteogenic cells. Thus, using dytical and preparative kwcytometry in combination with colony assays for bone and cartilage formation, 1 have demonstrateci that the responses of periosteal cells to rhOP-1, as a representative BMP, are dependent upon the diffefentiation state of the ceiIs as reflected in their size, cytoplannic granularity and OPN content- CHAPTER V

Intraceiiular OPN is an Integral Part of a CD44-ERM Complex Involved in Cell Migration ABSTRACT Osteopontin (OPN)produced by osteogenic cells is a prominent glycoprotein in the mineralized matrix of bone. However, cells in many other tissues dso express OPN. In previous studies an intracellular form of osteopontin with a peri-membranous distribution was identifid in migrating fetal fibroblasts. Since the presence of OPN in migrahg cells is consistent with the expression of OPN and CD44 by metastatic celis and by activated macrophages and lymphocytes, the relationship between the intracellular OPN and ce11 migration was studied using confocal microscopy to

analyze cells stained for OPN and CD44 by immunofluorescence. A distinct CO- Iocalization of the perimembranous OPN and the cell-surface CD44 was observed in fetal fibroblasts, periodontal ligament cells, activated macrophages and breast cancer cells. The CO-locdizationof OPN and CD44 was prominent at the leading edge of migrating fibroblasts, where OPN dso CO-localized with enin, as weil as in celi processes and at attachments sites of hyaluronan-coated beads. The subcortical location of the OPN in these cells was verified by cell-surface biotinylation experiments in which biotinylated CD44 and non-biotinylated OPN were isolated fiom complexes formed with hyaluronan-coated beads and identified by immunoblotting. It appears unlikely that the peri-mernbranous OPN represents secreted protein intemalized by endocytosis or phagocytosis, since exogenous OPN added to ce11 cultures could not be detected inside the cells. A physical association of OPN, CD44 and ePinlradixin/moesin (ERM), but not with vinculin or P-actin, was indicated fiom irnmunoadsorption and immunoblotting of ce11 proteins in complexes extracted kom hyaluronan-coated beads. The functional significance of OPN in this complex was demonstrated using OPN -/-ve fibroblasts 6om OPN-nul1 transgenic mice which displayed an impaired ability to migrate in a chernotactic gradient and reduced attachment to the hyaluronan-coated beads, but not to collagen-coated beads. Based on these studies I suggest that an intracellula. form of OPN is a.integral component of an actin-independent, hyaluronan-CD44-ERM attachent complex that is involved in the migration of fetal and metastatic cells and activated macrophages. INTRODUCTION Cellular adhesion and migration are fundamental processes required for morphogenesis in embryonic development and for wound healing in adult tissues (Gumbiner, 1996; Knudson and Knudson, 1993; Rauiger et al., 1997). These processes are dependent, in part, on the iniracellular re-arrangement of actin filaments and cytoskeletal-related proteins that are responsive to changes in the extracellular matrix environment. Communication between cytoskeletal proteins and the extracellular matrix is mediated through transmembrane receptors, some of which also participate in cell adhesion and migration (Dunlevy and Couchman, 1993; Gumbiner, 1996; Sherman et al., 1994). Hyaiuronan, a prominent component in matrix of embryonic tissues, influences ce11 behaviour during development and is also associated with the migratory behaviour and metastatic capacity of tumor celis (Ellis and Schor, 1996; Huang-Lee et al., 1994; Patel et al., 1995). In wound healing, which recapitulates the fundamental processes that occur in development, the presence of hyaluronan in the provisional matrix facilitates ceil migration (Savani et al., 1995), and in fetal tissues prornotes regeneration of co~ectivetissues (Mast et al., 1992). OPN, which is expressed early in development (Nomura et al., 1989), is also involved in ce11 attachment (Liaw et al., 1995), migration (Yue et ai., 1994)- chernotaxis, differentiation (Mark et al., 1987; Zohar et al., 1997b), malignant transformation (Craig et al., 1989; Senger et al., 1980) and immune ce11 activation (Patarca et al., 1993). The inductive effects of OPN on ce11 attachrnent and migration involve binding of extracellular OPN to integrin receptors (Bennett et ai., 1997; Kunicki et al., 1997; Liaw et al., 1995). The induction is enhanced by thrombin cleavage which increases the accessibility of the RGD integrin binding site (Senger and Pernizzi, 1996). However, not al1 OPN-related hctions are inhibited by RGD peptides, indicating that receptors other than integrins mediate OPN effects (Katagiri et al., 1996). Another trammembrane receptor that can bind to extracellular OPN is CD44 (Weber et al., 1996). Notably, both CD44 (Alaish et al., 1994; Bartolazzi et al., 1994; Hale et al., 1995; Nagabhushan et ai., 1996) and OPN (Chambers et al., 1996; Craig et al., 1989; Denhardt and Guo, 1993; Feng et al., 1995) are expressed in fetal tissues and by activateci immune cells and by malignant cells. Moreover, transfomation of benign epithelial -or cells by transfection with either OPN or CD44v can induce a metastatic phenotype (Chen et al., 1997; [ida and Bourguignon, 1997). CD44 was nrst recognized as a hyaluronan (HA) receptor. Ligation of CD44 receptors by HA can promote wound healing (Alaish et al., 1994), himor development and progression (Bartolazzi et al., 1994). CD44 expression is also associated with increased ce11 motility (Kaaijk et al., 1995; Sy et ai., 1996; Zhu and Bourguignon, 1996) whereas induction of apoptosis is associated with CD44 shedduig and the loss of motility (Gunthert et al., 1996). A large number (-100) of CD44 isofoms are generated by alternative splicing of transcripts coding for the proximal extracellular domain, which includes the hyaluronan binding site. Thus, hyaluronan binding is not a trait common to al1 CD44 isoforms. However, CD44 isoforms that bind hyaluronan augment the rapidity of tumor formation by melanoma cells in vivo (Bartolazzi et al., 1994), while transfection with a variant (vlO/exl4) of CD44 induces loss of adhesiveness to hyaluronan and increased migratory properties @da and Bourguignon, 1997). Hyaluronan also interacts with RHAMM, another transmembrane receptor (Entwistle et al., 1996). Ligation of RHAMM increases tyrosine phosphorylation of cytoskeletal-associated proteins such as p125fak and the proto-oncogene pp60 c-src (Hall and Turley, 1995). This results in an alteration in the turnover of focal adhesions and changes in ce11 motility (Hall et al., 1994). However, in contrast to RHAMM, no direct correlation between CD44 and the typical adhesion complexes associated with intracellular signaling pathways has been descnbed. Consequently, it has been suggested that CD44 binding by hyaluronan does not invoke signal transduction (Entwistle et ai., 1996; Koshiishi et al., 1994). The relationship between CD44 induction and increased ce11 motility may be mediated by putative actin-binding proteins such as the enin, radixin and moesin (ERM family) (Tsukita et al., 1994). This family of proteins is thought to Link cell- surface adhesion molecules to actin filaments in the subcortical area and thereby to serve as membrane cytoskeleton linkers. Moreover, ERM proteins may facilitate actin filament assembly and transduction of signals through tyrosine kinases or G-proteins (Berrymau et al., 1995; Takaishi et al., 1995) in fetal development, malignant transfomation, ce11 metastasis and immune responses (Haynes et al., 1991). Based on the apparent correlation between the expression of OPN and CD44 in developing tissues, and the enrichment of intracellular OPN in migrating cells 1 hypothesized that the perimembranous OPN, described in chapter II, may be associated with CD44 complexes involved in cell migration. Such an association would aiso be consistent with the CO-expressionof OPN and CD44 in metastatic cells and activated idammatory cells. The studies described in this chapter have utilized a combination of immunocytochemical, biochemical and fiuictional analyses to test this hypothesis. MATERIALS AND METHODS Ce11 Culture Rat dermai fibroblasts were derived hmskin explants removed fiom 21-day- old rat fetuses. Huma.penodontal ligament fibroblasts were obtained nom explants of tissue dissected fiom bicuspid teeth extracted fiom an 8-year-old male for orthodontie procedures. The perïodontal ligament was scraped fiom the apical part of the roots to avoid gingival fibroblast contamination. Both ce11 types were grown in a-MEM containing 15% heat-inactivated FBS and antibiotics (100 g/ml penicillin G, 50 g/d gentamycin sulphate, and 0.3 @ml fimgizone). For these studies, cells at passages 3 and 4 were grown as monolayers in T-75 flasks at 37OC in a humidified atmosphere of 95% air/5% C02. Cells were washed with PBS to remove non-viable celIs and then released with 0.01% trypsin in citrate buffer. Aiiquots were counted electronicaily (ZM Coulter Counter) and replated in 60 mm diameter culture dishes (Falcon, Becton Dickinson, Mississauga, ON) at a density of 7.5xIO4cells per dish. For attachent and migration assays, a fibroblastic ce11 line fiom an osteopontin-nul1 transgenic mouse fehis, (strain 129 C57BU6 F2, clone-135-3T3, provided by Dr. S. Rittling, Rutgers University, Piscataway, NJ) was compared to an embryonic mouse ce11 line NIH-3T3 which expresses OPN. For analysis of OPN-CD44CO-localization, human monocytes were isolated fiom fkesh blood sarnples and activated by incubation with 4 pg/ml concanavalin A (Con-A) for 3 h. In addition, human adenocarcinorna ce11 lines T-47D and MDA-MB-231 were obtained fiom the Amencan Type Culture Collection (ATCC). Cells were grown in a-MEM containbg 15% FCS and antibiotics. Immuno~ogica~Reagents Biotin-conjugated mouse anti-human CD44 monoclonal IgGl antibody (clone # A3D8), mouse anti-human vinculin monoclonal IgGl antibody (clone # hm-I), mouse anti-rat P-actin, monoclonal IgGl antibody (clone # AC-40), FITC-conjugated goat anti-mouse IgG antibody, FITC- conjugated rabbit anti-rat IgG antibody, TRITC- conjugated goat anti-mouse IgG antibody, Streptavidin-Quantum ~ed~~,Honeradish peroxidase-conjugated goat anti-mouse IgG, rabbit anti-rat IgG, rabbit anti-goat IgG, sheep anti-rabbit IgG antibodies and streptavidin were purchased fkom Sigma Chem. Co. (St. Louis, MO). Biotin-conjugated mouse anti-rat CD44 monoclonal IgG2a, (clone # 0x49) was purchased nom Phamringen (San Diego, CA). Mouse anti- human ezrin monoclonal IgGl antibody was purchased nom Transduction Laboratones (Lexington, KY). Five different antibodies to rat OPN were used in these studies; a mouse monoclonal anti-rat OPN antibody (MPIIIBIOI) was obtained fiom the Developmental Studies Hybridoma Bank (Johns Hopkins University, Baltimore, MD under contract from MCHD). Rabbit and goat polyclonal anti-rat bone OPN antibodies were fiom Dr. W.T. Butler (Dental Branch, University of Houston, TX); a rat monoclonal anti-human OPN antibody was provided by Dr. J.R. Hoyer (School of Medicine, University of Pemsylvania, Philadelph, PA) and an afnnity-purïfied rabbit anti-porcine OPN antibody was prepared in this laboratory (Zhang et al., 1990). Bead Attachent and Quantitarion Femc oxide microparticles (hereafter called "beads"; Aldrich Chemicals, Milwaukee, WI) were coated by incubation in solutions of hyaluronan (5 mg/ml), bovine collagen (3 mg/ml; Vitrogen; Celltrix Corp; CA) or bovine serum albumin (BSA; 5 mg/ml), as described in detail for collagen previously (Glogauer et al., 1995). Measurement of the distribution of bead diameters was performed by electronic particle counting (Coulter Channelyzer, Coulter Electronics, Hialeah, FL) and this showed a cross-sectional-area-weighted mean diameter of 4.5 p.Beads were rinsed in ca2'-~g-kePBS, washed three times and resuspended in PBS. Beads were added to attached cells, incubated for 20 min at 37OC, and the cells washed three tirnes with cold PBS to remove unbound beads. The surface area of dorsal ce11 membrane in contact with either hyaluronan, BSA or coilagen-coated beads was estimated by image analysis (Bioquant, R&M Biometrics; NashvilleJN). Cells plated on 8-well coverslip chamber slides (Nunc, Roskilde, Denmark) were incubated with beads (20 mg/ml) for 10 min and washed three thes with PBS to remove unbound beads or looseiy attached beads. Equal bead loading of cells in the hyaluronan, BSA and collagen-coated ce11 groups was verified by microscopy. Cells were stained with hematoxylin and eosin prior to anaiysis to facilitate identification of the ceil periphery for estimahg the percentage of the ce11 surface covered by beads. Isolation of HyaIuronan-Assonnted Compleres Proteins enriched in hyaluronan-bead adhesion complexes were isolated using the methods of Plopper and Ingber (1993). Briefly, ceils with attached beads were collected by scraping cells into 400 pl of 50 mM Tris-HCl, 10 mM CaC12, pH 8, containing 0.1 % Triton-X. The resultant cell-bead suspension was sonicated for 10 sec (output setting, 3, power 15%; Sonifier 185, Branson), and then homogenized in a 2 ml Dounce homogenizer (10 strokes). Beads with bound attachment complexes were isolated at 4OC using a side-puil magnetic isolation apparatus (Dynal, Lake Placid, NY; Fig V. 1). Mer rernoving the unbound ce11 hgments by washing 3 times with Tris buffer, the beads were re-suspended in immunoprecipitation buffer (LP; 0.3% voVvol Nonidet P-40, 0.3% wt/vol sodium deoxycholate, 0.15% Wvol BSA in distilled water, and 0.02% wt/vol sodium azide). Bead sarnples were heated at 9S°C for 5 min and re-sonicated for 10 sec pnor to protein analysis. Biotinyiation The procedures used to biotinylate cells and recover the hyaluronan-associated attachment complexes is summarized in Fig V. 1. Cells were surface labeled with sulfosuccinimidyl-6-(biotinamido)hexanoate (NHS-LC-Biotin; Pierce Chem. Co., Rockford, IL) following the supplier's protocol. Briefly, cultures were washed 3 times with ice-cold ca2-'-~g-fkeePBS, pH 8, and incubated with 0.5 mg/ml of NHS-LC- Biotin for 30 min at RT. Cultures were washed again 3 times with cold PBS and incubated with 500 pi (20 mg/ml) of hyaluronan-coated beads in PBS for 30 min at 37'C (Fig V.1A). Mer washing with coId PBS to remove unbound beads, the biotixïylated cells with attached beads were collected. Bead complexes, magnetically isolated as described above, (Fig V. 1B) were homogenized to ensure disintegraîion of ce11 membranes, re-suspended in immunoprecipitation buffer, heated at 95OC and sonicated twice for 10 sec (Fig V. 1C; output setting, 3, power 15%) to dissociate proteins bound to beads (Fig V. ID). The dissociated proteins were passed through a coiurnn of Sepharose-4B-streptavidin beads. @ Coated Bead \ Trammembrane Protein O Intracellular Protein Biotin

Collect Homogenizew w Magnet

Heat 95% Sonicate

Fig- V. 1- Analysis of Biotinylated CeII-surface Proteins. Rat derka1 fibroblasts were fvst Iabeled with biotin (A) and then hyaluronan-coated beads were allowed to attach to the ce11 surface. The cells were scraped, homogenized and the coated beads, with associated cell-surface components were magnetically isoiated (B). After washing the beads and separating them fiom ce11 debris (C) the biotinylated proteins and complexed non- biotinylated proteins were eluted by heating at 95'~and sonicating in immunoprecipitation buffer @). The unbound fhction hmthe column was colkcted and the column was washed with IS. buffii, foilowed by an elution with SDS-PAGE sample buffer to separate non- biotinylated proteins that were associated with biotinylated proteins bound to the column. The streptavidin-Sepharose beads were removed from the column and washed again with SDS-PAGE sample buffer to remove any rernainuig non-biotinylated proteins. Biotinylated proteins were eluted by incubating the streptavidin beads with 0. 1N HCl for 20 mui at room temperature (2 1°C). Immunoprecipitation and Western Blotting Protein samples were immunoprecipitated using antisera to human and rat CD44 or to rat OPN using a procedure described previously (Yao et al., 1994; Zohar et al., 1997). BriefIy, sample volumes were adjusted to 500 pl using LP. buffer and 200 pl aliquots were incubated with 5 pl normal rabbit sem for 2 h at 4OC, in 1.0 ml microfuge tubes. Pansorbin (100 pl; Calbiochem, San Diego, CA) was added and incubated for a further 2hat 4OC. Material that was non-specifically adsorbed to the pansorbin was rernoved by centrifugation (14,000 g for 7 min). The supernatant was transferred to a new microfuge tube and 4 y1 specific antibody conjugated to protein- A-Sepharose was added and incubated over-night at 4OC with gentle shakuig. The immune complexes were collected by centrifugation and the remahhg supernatant was either discarded or used for a second irnmunoprecipitation with a different antibody. The immune complexes were washed 5 times in PBS containing 0.5% Tween 20, dissociated by the addition of 40 pl SDS-PAGE sample buffer, containhg dithiothreitol, and heated for 5 min at 95'~.SDSPAGE electrophoresis of proteins was conducted using Tris-tricine gels, 10% T, 3% C as described in detail previously (Yao et al., 1994; Chapter II; Zohar et al., 1997). Proteins separated by SDS-PAGE were transferred to nitrocellulose membranes (0.45 m pore size) and immunoblotting was perfoxmed with the following prîrnary antibodies: mouse anti-rat OPN monoclonal antibody (diluted 1500); rat anti- human OPN monoclonal antibody (diluted 1200); rabbit or goat anti-rat OPN polyclonal antibodies (diluted 1 :100; 1:2000, respectively); mouse anti-human ezrh monoclonal antibody (diluted 1:250); mouse anti-human vinculin monoclonal antibody (diluted 1: 1000); biotinylated anti-CD44 monoclonal antibodies (mouse anti- rat, diluted 1: 125; mouse anti-human, diluted MO). The primary antibodies were detected with the following horseradish peroxidase-conjugated antibodies: anti-mouse (diluted 1:200), anti-rat (diluted 1:2000), anti-goat (diluted 1:5000), anti-rabbit (diluted 1: 1000) or with streptavidin (diluted I :L 500) as descnbed previously (Chapter II; Zohar et al., 1997). hunoreactive proteins were visualized by cherniluminescence detection using a commercial (ECL; Amersham, Life Science, Oakville, ON) followed by exposure of the blots to Kodak SB autoradiography film. Imrnunostaining and Confocal Microscopy Cells grown on covenlip chamber slides (Nunc, Roskilde, Denmark) were stained and examined with a 63X, 1.3 N.A. oil immersion objective using epifluorescent optics and confocal irnaging Ceica). Consecutive transverse optical sections were obtained fkom the level of celi attachent at the substratum to the dorsal surface of the cell. For antibody staining of OPN and CD44, human cells were washed 3 times with 0.25% BSA in cap-~g-fieePBS and incubated with anti-human CD44 antibodies diluted 1:40 in the BSA b&er for 1 h at 4OC. Cells were washed with the BSA solution and fixed by incubation in methanol at -20°C for 15 min. Cells were permeabilized in 0.1% Triton-X- 100 in PBS for 30 min at room temperature and incubated with primary antibodies for hurnan OPN diluted 1:40 for 1 h at 4OC. Cells were washed again with BSA bder aod incubated with a mixture of fluorescein- conjugated anti-rat antibodies and rhodamhe-conjugated streptavidin @oh at a 1: 100 final dilution). Samples were incubated for 30 min at 4OC followed by 15 min at roorn temperature. For hurnan periodontal cells stained for OPN and ezrin, cells were first fixed with methanol at -20°C for 15 min, permeabilized in 0.1% Triton-X-100 in PBS for 30 min at room temperature and incubated with a mixture of primary antibodies (OPN 1:40 and antienin 1:25 dilutions) for 2 h at 4OC. Slides were washed with BSA solution and incubated with a mixture of fluorescein-conjugated anri-mouse antibodies a? 1: 100 dilution and rhodamine-conjugated anti-rat antibodies at a 1:25 dilution. Cells were incubated for 45 min at 4OC followed by a 10 min incubation at room temperature. In some experiments human ceiis were stained with anti-OPN antibody foilowed by TRITC-phdoidin to compare the distniution of OPN with actin. The distnion of OPN in migrant and non-migrant ceiIs was analyzed in fetai rat fibroblasts separated in a Boyden chambers as descnied above and in chapter II (Zohar et al., 1997a). NB-3T3 and cells hmOPN null mice were also separated in Boyden chambers. Non-migrant ceils were removed fiom the upper membrane dace by scraping. The migrant celis and the lower surface were stained by incubating with propidiurn iodide (Sigma) 20 pglml in PBS for 30 min at RF and the number of migrating cells was assessed by image analysis as described by Hughes and McCulloch (1991). In order to avoid cross-reactiviv in rat dermal cells stained for OPN and CD44 due to the use of two primar- mouse monoclonal antibodies a modified staining protocol was used. Cells were first fixed with 2% paraformaldehyde in ~a"and ~g- fi-ee PBS for 30 min, washed 3 times with BSA buffer and permeabiiized by treatment with 0.1% Triton-X-100 in PBS for 30 min at room temperature. The cells were incubated with the prirnary antibody for rat OPN (diluted 1:800), washed again with BSA b&er and incubated with fluorescein-conjugated anti-mouse antibody (diluted 1:100) for 30 min at 4OC, followed by a 15 min at room temperature. Cells were washed again in BSA bder and incubated with biotin-conjugated anti-rat CD44 antibody (diluted 1: 1 25) for 1 h at 4OC, washed again with BS A buffer and incubated with rhodarnine-conjugated streptavidin (diluted 1: 1 00). Cells were washed with Ca2+- MC-fiee PBS and mounted. Controls for non-specific staining were perfonned in separate wells on the same slides using secondary antibody only. Coverslips were washed with PBS and mounted with an anti-fade rnounting medium (ICN). The spatial localization of OPN, CD44 epin and actin in bead/membrane complexes was imaged in single cells by confocal rnicroscopy. For FITC-labeled probes, excitation was set at 488 nrn and emission was collected with a 530/20 nm barrier filter. For TRITC and TexasRed, excitation was set at 530 nm and emission was collected at 620/40 nm. Analysk of Intracellluar OPN To investigate whether the intracellular perimembranous fom of OPN (Zohar et al., 1997) is the result of intemalization of extraceilular OPN, two sets of expeeents were done. In the firsf mouse NM-3T3 fibroblasts, mouse OPN-/- and rat fetal dermal cells were grown on coverslip chamber slides. On day 4, cells were washed twice with a-MEM and re-fed with a-MEM containing 5% FBS for 6 h and then incubated with purified porcine OPN (2 pg/ml) for 24 h. Cultures were washed, futed, pemeabilized and stained with rabbit anti-porcine OPN antibody. In a second series of experiments NM-3T3 and OPN-/-fetal fibroblasts were grown in T-25 flasks for 6 days, and incubated for 24 h with conditioned medium from FRCCs cultures, which has been shown to secrete abundant OPN (Zohar et al., 1997a). Cells were trypsinized, stained with the monoclonal anti-rat OPN and analyzed by flow cytometry as descnbed in Chapter II (Zohar et al., 1997a).

RESULTS Perimembranouî OPN and Cell Migration Previous analyses of fetal rat cells have revealed two distinct patterns of intracellular OPN with an enrichment of intracellular OPN in migrating cells [Chapter II; Zohar et al., 1997al. To characterize the intracellular pattern of OPN distribution in migrating and non-migrating cells, fetd rat demial fibroblasts were separated in a modified Boyden chamber. Filters containing the migrating and non-migrating demal fibroblasts were cut to size and stained with the monoclonal anti-rat OPN antibody and mounted so that both cell populations were analyzed on the same slides. In non- migrating cells, perinuclear staining with a punctate cytosolic pattern of OPN, typical of secreted proteins, was predominant; perimembranous staining was also identified in a small minority of cells (Fig V.2). In contrast, migrating cells exhibited perimembranous staining, with no apparent perinuclear staining (Fig V.2). Notabl y, the perimembranous staining was O fien concentrated in filopodia-like structures. The unique subcortical distribution of intracellular OPN in motile fetal cells was examined for a possible functional Iink to ce11 attachment capacity which is 124 associated with cell traction (Mitchison and Cramer, 1996). Femc oxide microbeads were coated with type4 coliagen which is known to interact with P-integrin receptors (Fadeabas et al., 1997), or with hyaluronan, which has an important role in fetal ce11 migration (Estes et al., 1993; Monnet-Tschudi et al., 1993). BSA-coated beads served as a control. To relate OPN expression to the adhesive properties of cells, fetal mouse OPN-/- cells were compared with fetal mouse (NIH-3T3) OPN+ve cells for their capacity to attach to coated microparticles. The OPN-/- cells exhibited the same adhesive capacity to type-1 collagen- or BSA-coated beads as the OPN+ve cells (Fig V. 3A) when measured by the percentage area of the ce11 covered by the beads. In con- hyaluronan-coated beads covered only 10-15% of OPN-/- cells but covered 50 % of the OPN+ve NTH-3T3 cells. When subjected to a chernotactic gradient in a Boyden chamber, the migration of the OPN-/-ve cells was significantly @< 0.001) reduced compared to NIH-3T3 cells (Fig 3B). To characterize the proteins associated with hyaluronan in this attachent complex, rat dermal fibroblasts were first incubated with hyaluronan-coated beads, and the beads were then magnetically isolated fkom the cells (Plopper and Ingber, 1993). The cell proteins atiached to the beads were eluted and analyzed on Western blots for OPN and also for CD44 which is the main receptor for hyaluronan in fetal tissues (Adolph et al., 1993; Zhang et al., 1996). AIthough several bands correspondhg to the various isoforms of CD44 were observed in a total ce11 extract(1ane l), the 80 kDa isofom was emiched in the bead extract (lane 2). Immunoblotting for OPN revealed a protein doublet with the major band at 72 kDa (Fig V. 4A). Hyaluronan Anachment Complexes and Iniracellular OPN Since OPN has been show to act as a ligand for CD44 in migrahg cells (Weber et al., 1996), the affinity of intracellular OPN to hyaluronan-CD44 complexes was compared to BSA-coated bead extracts and whole cell lysates. Samples were bt immunoprecipitated with the anti-rat CD44 antibody and analyzed by immunoblotting. When probed with the monoclonal anti-rat OPN antibodies and quantified by Fig V '. 2- Discrete Immunocytochemical Staining Patterns for Intracelluiar Osteopontin- Confocal fluorescence micrographs of migrating and non-migrating fetal dermai fibroblasts separated in a Boyden chamber and stained for OPN using a mouse anti-rat OPN monoclonal antibody and a FRC-Iabeled rabbit anti-mouse igG second antibody. Migrating cells exhiiited a perirnembranous pattem of OPN staining dong the cell membrane and ceU processes, with the OPN concentrated in focal deposits. Non-migrating celis exhibited a punctate perinuclear staining pattern, typical of secreted proteins, with extraceUular immunoreactive materid also present on the nitroceLlulose membrane. HA Co1lagen-I BSA

Fig V. 3- Migration and Binding of Coated Beads to OPN-1- and OPN+ve Embryonic Mouse Fibroblasts. A- Binding to coated beads. Fibroblasts €rom transgenic OPN-null mice and NM-3T3 (OPN+ve) ceiis were incubated with equivdent numbers of magnetic beads coated with hyaluronan, BSA or type 1 coiiageo. Mer washing to remove unbound beads, the percentage area (mean 9s.e.m) of the cells covered by beads was determined by image andysis. Whereas binding to type 1 collagen and non-specific biadiag to BSA was not significantly different between the two celi types, the OPN-ve celis exhiIbited a -3 fold lower capacity to atîach to hyaluronan-coated beads (p< 0.00 1) compared to the MH-3T3 ce&. B- Ceil migration in a Boyden chamber. The same cek were plated on gelatin-coated membranes in individual weils of a mdti-weil Boyden chamber and subjected to a chemotactic gradient formed by addition of 15% concentrations of fetal caif senun added to the chambers. Non-migrating cells were removed by scraping. Celis migrating through the membrane into the lower chamber were stained and counted using an image analyzer. The resuIts are averaged fiom two independent assays, each performed in îriplicate. densitomeûy, hyaluronan-bead samples exhibited -1 O-fold more OPN than the BSA- bead samples, with a Cfold enrichment in relation to whole ce11 extracts (Fig V. 4B). The same membrane was stripped and probed with a rabbit polyclonal anti-rat OPN antibody, which confïrmed the enrichment of OPN in hyaluronan complexes associated with CD44 (Fig V. 4B). In addition, the CD44 immunoprecipitated samples were also probed for two other subcortîcal intracellular proteins, vinculin and ezrin, both of which were present in the rat hyaluronan-CD44-OPN complexes (data not shown). However, only ezrin was enriched in hyaluronan extracts compared to the BSA extracts. To investigate the relationship between intracellular OPN, ceil surface OPN and the isolated hyaluronan attachment complexes, celî-surface proteins of fetal rat dermal cells were labeled with biotin. Proteins eluted fiom hyaluronan-beads were passed through the streptavidin-Sepharose column to capture the biotinylated protehs. The unbound, non-biotinylated proteins that passed through the column (S) were compared to the biotinylated proteins (B), released fiom the streptavidin-Sepharose beads with O.LN HCl and whole dermal ce11 extracts 0).Samples were analyzed for OPN on immuno-blots using an anti-rat OPN monoclonal antibody and polyclonal goat anti-rat OPN antibodies (Fig V. SA) which revealed an immunoreactive band in the non-biotinylated fraction (S), and only a trace in the biotinylated hction (B). The immunoreactive bands CO-rnigratedwith OPN in an EDTA extract prepared kom FRCC bone nodules (E). Biothylated proteins in the hyaluronan-bead extracts and biotinylated proteins eluted nom the streptavidin-Sepharose column were immmoprecipitated using anti-rat CD44 antibodies. Unbound proteins from the CD44 imrnunoprecipitation were dso subjected to a second immunoprecipitation using anti-rat OPN antibodies. Analysis of proteins on immuno-blots using streptavidin-peroxidase to identiQ biotinylated proteins revealed an 80 kDa biotinylated protein in both the whole bead extract and in the hction isolated fforn the streptavidin-Sepharose beads (Fig V. SB). 72> OPN

Fig V. 4- Western Blot Analysis of OPN and CD44 Extracted from Rat Dermal Fibroblasts. A- Proteins kom whole-cell extracts (lane 1) and extracts of hyaluronan-coated beads (lane 2) were separated by SDS-PAGEand transferred to a nitrocellulose membrane. The membrane was fmt probed with a biotinytated mouse anti-rat CD44 antiiody, which recognizes a domain common to most of the CD44 isofom (upper panel). Four bands were identified in the ce11 extract, ranging between 50-210 kDa, whereas only the 80 kDa band was identified in the hyaluronan bead extract. After stripping, the membrane was probed with a mouse anti-rat OPN antibody which revealed two cIosely-spaced bands, in both the ceIl and bead extracts, correspondhg in size to rat OPN isofom. B- Proteins in complexes immunoprecipitated with mouse anti-rat CD44 antibody from ce11 extracts, hyaluronan-coated and BSA-coated bead extracts (lanes 1, 2, 3 respectively) were separated by SDS-PAGEand transferred to a nitrocellulose membrane. The blot was probed first with a mouse monoclonal antiiody to rat OPN (upper blot) and, after stripping the membrane, probed with a rabbit polyclonal antibody for rat OPN (lower blot) to confi the presence of OPN and the enrichment of OPN in CD44 complexes obtained from the hyaluronan bead extracts. Fig V. 5 - Anaiysis of Biotinyiated Ceil-surface Proteins on Western Blots. Foilowuig ceil-surface biotinyiation, fetal rat dennai fibroblasts were incubated with hyaluronan- beads and the beads, with the associated attachment proteins, isolated as desrnid in Fig V. 1 After separating the proteins fiom the beads the biotinylated proteins (B) were selectively bound to a streptavidin-Sepharose affinity resin, A-The non-biotinylated proteins (S), that did not bind to the streptavadin-Sepharose, and the biotinylated proteins released fiom the streptavidin-Sepharose beads (B) were anaiyzed for OPN on inmuno-blots and compared to OPN detected in whole dermai ceU extracts @) and in a 0.5M EDTA extract of mineralized boue nodules produced by FRCCs in cuIture (E). The blot was probed with a polyclonal goat anti-rat OPN antiody which indicated the presence of an immunoreactive band in the whole ceU extracts and the non-biotinylated (S) bction that co- migrated with an OPN band in the EDTA extract, with oniy a trace of OPN in one of the duplicate lanes of the biotinylated material (B). B, C- The hyaluronan bead extracs and the biotiuyIated proteins released fiom the streptavidin- beads were immunoprecipitated 6rst with anti-CD44 antiidies (lanes 2 and 5, respectively), foilowed by anti-OPN antiiodies (lanes 3 and 6, respectively). The unbound material fiom the two immunoprecipitations (lanes 1 and 4 respectively) were analyzed by immunoblotting. When probed with sîreptavidin-HRP strong bands, representing biotinylated proteins that were not imrnunoadsorbed by the antiiodies, were observed in Ianes 1 and 4 while a single band of -80 kDa was detected in Ianes 2,3 and 5. Stripping and re-probing with the anti-rat OPN anniody showed a band in the CD44 immunoprecipitation (lanes 2 and 5) and in the OPN immunoprecipitation of the hyduronau bead extract (Iane 3). A biotinylated protein (lane 3; Fig V. 5B) was identifieci in the hyaluronan complex imrnunoprecipitated by the anti-rat OPN antibodies. This band, which could represent biotinylated OPN bond in the hyalmnan bead complex, more Likeiy represents biotinylated CD44 bond to OPN, since biotinylated OPN was not detected in the proteins isolated f?om the streptavidin beads (lane 6; Fig V. SB). To determine the presence of OPN in the hyaluronan-bead extracts and in the CD44 immunoprecipitations, the membranes were stripped and reprobed with the anti-OPN antibody (Fig V. 5C). Immunoreactive bands indicating the presence of OPN were present in the CD44 and OPN immunoprecipitations of protein in the hyaiuronan bead extract. However, in the bioîinylated protein hction dissociated fkom the streptavidin column an immunoreactive band for OPN was observed in the CD44 immunoprecipitation, but could not be detected in the OPN immunoprecipitation, indicating that al1 the OPN was associated with the CD44 1 investigated the possibility that extracellular OPN had been intemalized by incubating purified porcine OPN (2 pgM) with mouse fetal OPN-/- and OPN+ve cells. Cells were then immunostained with an ad-porcine OPN antibody that does not recognize mouse OPN. No significant stalliing for porcine OPN was seen in any of the ce11 types studied. However, when replicate cells were immunostained with the anti-rat OPN antibody which recognizes mouse OPN, the OPN+ve cells exhibited perimembranous and perinuclear OPN staining. In a second experiment OPN-/-ve cells were incubated for 24hwith conditioned medium containhg OPN nom either NIH- 3T3 or FRCC cultures and intracellular OPN assessed using single ce11 flow-cytometry analysis to detect cells labeled with the anti-rat OPN monoclonal antibody that also cross-reacts with mouse OPN. FRCC and MH-3T3 exhibit mean OPN fluorescence of 180 + 23 (charnel numbers t s.e.m.), the mean fluorescence for the OPN-ve cells was 54 f 7 and for OPN-ve cells incubated with FRCC conditioned medium for 24 h the fluorescence was 35 + 1. Fig V. 6- Locdization of CD44 and OPN in Relation to Hyaluronan-coated Beads. Confocal and transmission Light rnicroscopy were used to analyze the imrnuno-fluorescence labeiing for CD44 and OPN, and for the location of hyaluronan beads on the surface of human periodontal ligament cells, The fluorescence for CD44 was c~IocaIizedwith OPN throughout the ce11 with foci of fluorescence associated with (either surroundhg or co-localized depending on the opticai section) hyaluronan-coated beads (mows). Fig V. 7- Western Blot Analysis of Whole Ceii and Bead Extracts from Human Periodontal Ligament Ceils. Whole ceii emcts, and extracts of hyaluronan-coated and BSA-coated beads incubated with human periodontal ligament ce& (lanes 1, 2 and 3, respectively) were immunoprecipitated with anti-human CD44 antiiody and the immuno-complexes separated by SDS-PAGE and transferred to a nitroceiiulose membrane. A- Membrane probed with a rat anti-human OPN antiiody, showing an enrichment for OPN in the hydwonan extract. An aliquot fiom the hyaiuronan extract was also digested with thrombin (lane 4) resulting in the Ioss of the 72 kDa band and the appearance of an immuno- reactive 40 kDa Çagrnent. B- Membrane probed with a biotinylated mouse anti-human CD44 antibody confinning the immunoprecipitation of the major 80 kDa isofonn of CD44 in all of the extracts with an eurichment in the hyduronan-bead extract compared to BSA-bead extract. C- Membrane probed with a mouse anti-human ePin antiiody showing an enrichment of ePin in the hyaluronan-bead extract relative to îhe BSA-bead. D- Membrane probed with a mouse anti-human vinculin antiiody exhibitmg a minor enrichment (-125%) in the hyaluronan-bead extract compared to the BSA-bead extract Spatial Distribution of CD44 and OPN The spatial distribution of OPN and CD44 in relation to the hyaluronan beads was imaged in single cells by confocal rnicroscopy. The cells were incubated with hyaluronan-coated beads, hed, permeabilized and then stained sequentially for OPN and CD44 The OPN and CD44 were CO-distributedin ce11 processes and dong the ceil membrane. Occasionally, foci of intracellular OPN and extracellular CD44 staining approximated the hyaluronan beads (Fig V. 6). Characterization of Intrrrcelltilar OPN Related Complexes in Human Periodontal Cells To substantiate the association of intracelluiar OPN with CD44 and hyaluronan-coated beads observed in fetal rodent stroma1 tissues, studies were also performed using human periodontal ligament fibroblasts incubated with hyaluronan- coated beads. The hyalmnan bead extracts were immunoprecipitated using a monoclonal anti-human CD44 antibody and probed with monoclonal antibodies to human OPN and CD44 (Fig V. 7A, B). The blots were also probed for ERM proteins that are known to associate with the intracellular domain of CD44 (Tsukita et al., 1994) using a monoclonal antibody to human enin, and for the focal adhesion protein vinculin (Dunlevy and Couchman, 1993) with a monoclonal anti-human vinculin antibody (Fig V. 7C, D). Hyaluronan extracts probed for human OPN exhibited >7 fold enrichment compared to the BSA extract (lanes 1 & 3, Fig V. 7A). When the immunoprecipitated hyaluronan extract was digested with thrornbin to cleave OPN, the 72 kDa band was lost and a 40 kDa OPN fkagrnent was generated (lane 4, Fig V. 7A). Probing the sarne membrane with CD44 antibodies showed a 2.5-fold enrichment for CD44 in hyaluronan complexes compared to BSA controls, but was -4 fold lower than the ce11 extract (lanes 2, & 3, respectively, Fig V. 7B). Probing for the ERM proteins exhibited Cfold e~chrnentin the hyaluronan extract compared to BSA, with lower levels observed in the total cell extract (Fig V. 7C). Vinculin was emiched only 1.3-fold in the hyaluronan extracts cornpared to BSA with comparable levels in the total cell extract (Fig V. 7D). CD44-OPN and Actin Distrrtrrbution To investigate a possible Mage between the hyaluronan-bead complexes and the actin cytoskeleton, imrnunoprecipitation of CD44 ftom rat derrnal fibroblast ce11 extracts obtained hm type-1 coliagen-coated beads, hyaluronan-coated beads and BSA-coated beads were analyzed on Western blots for the presence of the main actin isoform (p-actin) using a monoclonal mouse anti-6 actin antibody (Fig V. 8A). Ceil extracts from type4 coliagen-coated beads exhibited Cfold e~chrnentfor p-actin compared to BSA-coated beads (lanes 3 and 4 respectively, Fig 8A), but no immunoreactive band for P-actin was detected in the hyaluronan-bead extract (lane 2). Human periodontal ligament cells were also incubated with hyaluronan-coated beads and stained for OPN followed by TRUC-phalloiciin to compare the distribution of OPN and actin. Confocal analyses of cells exhibiting lamellipodia-like extensions revealed OPN staining in focal sites that CO-localizedwith the hyaluronan-coated beads in ce11 extensions. However, a spatial association of actin filaments with hyaluronan-beads or OPN was not apparent (Fig V. 8B). Spatial Relutionship Between Hyaluronan, Intracellular OPN. CD44 and EN The spatial distribution of intracellular OPN, CD44 and ERM proteins was imaged in single human periodontal Ligament cells by confocal microscopy in transverse 0.5 pm optical sections. CD44 staining, which was done prior to ce11 fixation and permeabilization, showed a difise pattern of staining dong ce11 membranes. However, higher intensity CD44 staining was observed around focal sites of OPN, which were prominent in larnellipodia and at the leading edges of cells (Fig V. 9A). Staining for ERM proteins revealed a distinct CO-localizationof the protein with the OPN in the leading edges of cells (Fig V. 9B). Co-localization of OPN-CD44 was also observed in activated human macrophages and in two metastatic adenocarcinorna ce11 lines (Fig V. 9C) Fig V. 8- Relationship of Actin to the Hyaluronan-bead Complex. A-Western blot analysis for P-actin usïng a mouse anti-rat-actin antibody in sarnples of a rat dermal fibroblast ce11 extract (lane I), a hyaiuronan-bead extract (lane 2). a type4 collagen- bead extract (lane 3) and a control BSA-coated bead extract (lane 4). A band for actin was observed in each lane except for the lane with the hyaluronan-bead extract. Notably, the actin band was also absent in two replicate samples of hyaIuronan-bead extracts even at higher sample Ioading. B- ConfocaI fluorescence rnicrographs through the rniddle of a migrating human periodontal fibroblast, the leading edge attached to hyaluronan-coated beads. Cells were stained for OPN Iabeled with FlTC and for actin using TRITC-phahidin. Actin filaments did not CO-localize with the hyaluronan-beads nor with the OPN associated with the beads, although al1 of these components were present at the leading edge of the cell. Fig V. 9 A$- Confocal Fluorescence Microscopy of CD44 OPN and b. Human periodontal ligament fibroblasts were immunostained with moue anti-human CD44 antiiodies, rat anti-human OPN ant~iodiesand moue anti-buman ePin antibodies. A diaise staining pattern for CD44 was observed with a higher intwsity of staining around focal deposits of OPN (mer panels). Staining for ezrin and OPN (lower panels) reveded a distinct CO-localization of the protein with the OPN Ieading edges of the ceil. in the paneis on the right side the superimposition of fluorescence for the hwo different antiiodies demonstrates the co- locaiization of the proteins. C- Immimostaining for OPN and CD44 in macrophages and metastatic cek. Activated human macrophages (top panels) and in two metastatic epitheiial cell hes (T-47D and MDA-MB- 231; middle and lower panels, respectively) were stained consecutively for OPN (left-side paneis and CD44 (rigfit-side panels). Co-locaiization of OPN and CD44 dong the ceU membranes and in focal deposits dong celi membranes was observed for both ce11 types. 137 DISCUSSION These studies have identified an intracelMar fonn of OPN that is associated with a hyaluronan-CDWERM complex in migrahg embryonic fibroblasts. Co- localization of OPN with CD44 in activated macrophages and metastatic adenocarcinorna cells indicate that the complex may also be involved in mediating the migration of extravasahg cells and cancer celis, a hding that is consistent with the up-regulation of CD44 (DeGrendele et al., 1997; Naot et al., 1997) and OPN in these cells (Behrend et al., 1994; Chellaiah et al., 1996; Patarca et al., 1993). That OPN has a fiuictional role in the cornplex is demonstrated by the impaired ability of OPN-nul1 (OPN-/-ve) fibroblasts to bind to hyaluronan-coated beads and to migrate in a chemotactic gradient. Identzj?cation of Intracellular OPN Since OPN has been characterized as an extracellular protein produced by a variety of ce11 types (Bayless et ai., 1997; Chambers et al., 1996; Denhardt and Guo, 1993), its presence inside the ce11 in perimembranous foci, unrelated to secretory granules, was surprising. To ven@ that this immunostaining pattern, which was predominant in migrating cells, did indeed represent OPN, four different antibodies, including two monoclonai antibodies, were used and the immunocytochemical results conbed its intracellular location in various cells fiom rodent and human tissues. In addition, the same antibodies were used in Western blots to identify OPN in hyaluronan-associated complexes, and in CD44-associated complexes isolated by imrnunoprecipitation. Ln each case a prominent 72 kDa band, corresponding in size to OPN isolated fkom bone rnatrix, was identified on the immunoblots. Moreover, the band obtained from human periodontal ligament cells was cleaved by thrombin, producing a 40 kDa imrnuno-reactive kgment, consistent with previous studies demonstrating a tbrombh cleavage site in the central region of the osteopontin molecule (Senger et al., 198 9). Occasionally the imrnuno-reactive band was detected as a closely spaced doublet, as observed with secreted forms of OPN that are generated by differences in post-translational modifications (Nagata et al., 1991). In recent studies OPN has been shown to be a ligand for CD44, binding to the extracellular domain of the transmembrane pro tein and mediahg chernotactic migration (Weber et al., 1996). However, the perimembranous OPN observed in my studies does not appear to resuit hmthe intemalization of ce11 surface-bound protein, as indicated by cell-surface biotinylation experiments. Notably, biotinylated OPN could not be detected in hyaluronan-bead complexes or in CD44 complexes obtained by immunoprecipitation. Further, since the binding of hyaluronan and extracellular OPN to CD44 are mutuaily exclusive (Weber et al., 1997), the OPN enriched in the hyaluronan-bound complexes would not be expected to be cell-surface bond. hternaiization of OPN by a phagocytic mechanisrn also seems unlikely, as exogenous OPN, added to the culture media of fetal mouse fibroblasts, couid not be detected intraceI1ularly. Thus, the intracellular OPN associated with the CD44 attachment complex appears to be synthesized independently of secreted OPN, as indicated by the absence of perinuclear staining in migrant cells separated in a Boyden chamber. OPN and Hyaiuronan-CD44Attachrnent CompIexes Ligation of CD44 receptors by hyaluronan promotes ce11 adhesion and aggregation (Culty et al., 1990; Turley, 1992), and can promote tumor cell development and progression (Bartolaui et al., 1994; Goebeler et al., 19%). The cytoplasmic domain of CD44 is believed to interact with ERM proteins and am which may link the attachent complex to the cytoskeleton (Kalornins and Bourguignon, 1988). However a dennitive linkage to the actin cytoskeleton is questionable (Shuster and Herman, 1995) since non-physiologicd buffers were used in the association experiments. Although an ankyrin-like protein has also been identified as the cytoskeletal linkage for CD44 (Lokeshwar and Bourguignon, 1992), recent studies have failed to show ankynn as a component of CD44-ERM complexes isolated by irnmunoprecipitation (Tsukita et al., 1994). However, an unidentified 72 kDa protein observed in this complex could have been OPN, since OPN has a rnolecular weight of 72 k on SDS-PAGE and as 1 have demonstrated in this study OPN CO- localizes with ezrin at the leading edge of migrating cells. Vinculin, a protein that associates with actin stress fibers (Hall et ai., 1994) and which is present in classical focal adhesions complexes (Lauffmburger and Horwitz, 1996), was e~chedonly rnargindy in the hyduronan-CD44 complexes. Since vinculin did not CO-localizewith the perimembranous OPN, it does not appear to be a cytoskeletai Mage protein for this attachent complex. B-actin is one of the major isoforms of actin found in integrin complexes (Hynes, 1992) but was not detectable in the hyaluronan-CD44 complex. Moreover, confocal analyses using hyaluronan-coated beads, TRITC-phalIoidin and OPN antibodies did not show any association between actin filaments and either hyaluronan-beads or OPN. Although different types of focal contacts and adhesion complexes have been identified by interference reflection microscopy (Abercrombie and Dunn, 1975), attachment complexes lacking actin filaments or accumulation of vinculin have not been described previously. In contrast to other attachment complexes, signaling through hyaluronan-CD44 receptors has not been clearly documented, although signahg through the ERM proteins has been suggested (Berryman et al., 1995; Tsukita and Yonemura, 1997). In cornparison, a second hyaluronan receptor RHAMM (receptor for hyaluronate- mediated motility; (Turley, 1992)) has both signaling capacity involving tyrosine phosphorylations, activated src-kinase (Hall et al., 1996) and has an association with vinculin and actin stress fibers (Hall et al., 1994) that conforms to the classical type of focal adhesions seen in cultured fibroblasts (Hynes, 1992; Lauffenburger and Horwitz, 1996). However, since the RHAMM receptor may associate with other receptors, including integrins (E.Turley, personal communication), it is yet to be established whether the signalling observed is mediated directly through E2HAMM ligation alone or through an associated attachment complex. Similady, it is conceivable that the interaction with vinculin and actin may involve associated complexes since the RHAMM receptor does not have a cytoplasmic domain. Based on the novel properties of OPN that 1 have demonstrated in these studies, I have proposed a mode1 for the CD44 attachment complex in which the intracellular OPN acts as a structural, ligation unit required for the assembly of the complex and its functional association with signaiing pathways that cm effect ce11 migration (see Fig VI. 1; Chapter VI). 1 suggest that intracellular OPN may be involved in a localized restriction mechanism in which hyaluronan-ligated CD44 receptors aggregate; the ligated CD44 binding to the ERM proteins at sites of focal OPN accumulation- This suggestion is supported by the enhanced staining of ERM proteins in the vicinity of hyaluronancoated beads and intracellular OPN, even though the distribution of CD44 staining is relatively uniform dong the ce11 membrane. Thus one conceivable sequence may involve: 1) hyaluronan binding to CD44; 2) aggregation of CD44; 3) recruitrnent of OPN to a protein complex that includes the intracellular domain of CD44; and 4) the assembly of the ERM proteins into this complex. The multiple phosphorylation sites on OPN a? serine and tyrosine residues and its ability to bind specific protein motifs supports the notion that if OPN regulates the assembly of the complex, the reversible phosphorylation of intracellular OPN isofoms may be required. Further, recombinant OPN exhibits ectokinase activity (Ashkar et al., 1993). It is well recognized that the assembly of other adhesion complexes is dependent on the phosphorylation of proteins in attachent complexes including talin, paxillin and p125fak (Bolton et al., 1997; Dunlevy and Couchman, 1993; Kneg and Hunter, 1992). By analogy 1 suggest that the phosphorylation of OPN and its capacity to phosphorylate other proteins (e-g. eh) in the complex may provide a similar type of regulation. A more detailed discussion of this proposal follows in the next chapter. My demonstration of an intracellular form of OPN that associates with CD44 attachrnent complexes and which is required for ce11 migration and binduig to hyaluronan has provided insights into a unique location and role for OPN that is exhibited by fetal, metastatic and activated inflammatory cells. Al1 of these ce11 types are characterized by rapid motility in-vivo and by the relatively high proportion of migrating cells in the whole ce11 population of these tissues. The absence of a direct linkage between the OPN-CD44 attachent complex and the actin cytoskeleton is notable in this context, as rapid ce11 migration is not usually associated with well- developed and heavily cross-Wed actin filaments. In contrast, the assembly of actin filaments and other actin-binding proteins such as tropomyosin, a-actinin and nebulin cells. Thus the novel kding of an actin-independent attachent complex that is prominent in rapidly migrating cells may describe the moiety described previously as "close contacts" in interference reflection microscopy (Abercrombie and Dunn, 1975). Future work should be focused in examùiing the hctional relationship between close contacts and the CD44-OPN complexes that I have described in this chapter. CHAPTER VI

DISCUSSION Single ce11 anaiysis of intracelMar OPN has revealed two distinct phenotypes that relate to the differentiation of osteogenic cells and to the migration of embryonic strornal cells. I have shown that the presence of intracellular OPN in conjunction with morphological criteria and cellsycle aaalysis can be used to identify stages in the differentiation of osteogenic cells (Zohar et al., 1997). Moreover, in studies using cytokines that affect osteogenic differentiation in definecl ways 1 have confimied the utility of intracellular OPN combined with morphologicd criteria as an analytical tool for study of differentiation stages (Zohar et al., 1998~).The absence of OPN expression in the same osteogenic populations was used to characterize stem cells that were enriched within a population of small quiescent cells, providing the first direct evidence for such cells in stromal tissues (Zohar et al., 199%). The identification of a novel perimembranous pattern of OPN staining that is typically observed in migrating stromal cells, and which is associated with CD44 in activated macrophages and metastatic cells has uncovered a potentially unique role for OPN as an integral component of the HA-CD44-ERM attachment complex.

OPN and Intracellzrlar OPN OPN is synthesized as a 33kDa protein that has anionic properties due to the high proportion of acidic arnino acids. Within the primary amino acid sequence, polyaspartate and RGD motifs for binding to hydroxyapatite crystals and integrins respectively, indicate fundamental roles for OPN in regulating mineralization and mediating cell signalling and attachment. Phosphorylation of OPN at serlthr sites, and possibly at tyr sites as indicated by a PEST motif (Craig et ai., 1989), as well as autophosphorylating activity of recombinant OPN (Ashkar et al., 1993), suggest that OPN cm have a marked influence on ce11 signaling and attachent (Ashkar et al., 1993; Nemir et al., 1989; Salih et al., 1996). Phosphorylation can also enhance the ability of OPN to bind calcium ions (Mark et al., 1988; Singh et al., 1993). Glycosylation of OPN through N and O-linked attachent sites can influence the ability of OPN to bind to fibronectin and the ce11 surface (Nernir et al., 1989; Singh et al., 1990). Thus, a non- phosphorylated, N-glycosylated form of OPN (np69) binds fibronectin in solution whereas the phosphorylated protein @p69) associates with fibronectin on the ce11 surface. Moreover, the ceil-Surface binding of the pp69 requires sialic acid to be present in the carbohydrate side chahs (Shanmugam et al., 1997). The wide potential variability in post-translational modifications has been manifest in the expression of different fom of OPN in different tissues (Sodek et ai., 1999, including bone (Nagata et al., 1991), as well as in transfomed cells (Behrend et al., 1994; Craig et al., 1988; Shanmugam et ai., 1997). In studies of OPN expression during osteogenic differentiation, low and high phosphorylated and sulphated forms of OPN have been identified that relate to early and later stages of differentiation, respectively (Nagata et al., 1989). Although 1 found some selectivity for these different foms of OPN when using the monoclonal and polyclonal antibodies to rat OPN, neither antibody provided sufficient discrimination to allow a meaningfûl cornparison by Bow cytometry. In addition to the variability that is generated through the extensive post-translational modifications of OPN, different foms of OPN can dso be generated by aiternate splicing which occurs within the 5'- untranslated region of rat OPN (Singh et al., 1992) and within the open reading fhme (Crivello and Delvin, 1992; Saavedra et al., 1995). However, no splice variants involvuig the coding sequence of OPN have been isolated. Although OPN is expressed by a variety of cells, in each case it has been characterized as a secreted protein and the secreted protein has been implicated in either the formation of extracellular matrices, in regulating mineral crystal formation, or in ce11 attachent and migration. Thus, at the outset of my studies, which employed flow cytometry, 1 assumed that the intracellular OPN measured was being produced for subsequent secretion. Later analyses of migrating cells in which a peri-membranous staining pattern for OPN was observed with little or no perinuclear staining indicated that an intracellular fom of OPN was also present. Although studies designed to address the origin of this OPN were undertaken and were found to support its intracellular location, the metabolism of the pen-membranous OPN and its biosynthetic pathway have yet to be established. Nevertheless, in flow cytornetry analyses of intracellular OPN these diEit fom of intraceUular OPN are not distinguishable, necessitating single ce11 and combinatorid analyses to establish correlations with osteogenic differentiation.

OPN in Osteogenic Dz-tiution Previous studies of osteogenic cell cultures have revealed a biphasic pattern of OPN expression in which a minor and major peak in OPN mRNA is obsewed during the proliferative and matrix-formhg stages, respectively cian and Stein, 1992; Yao et ai., 1994). While the early peak in expression of OPN in periosteal ceils is especially low and may include expression by osteogenic and non-osteogenic migrant cells, in osteogenic bone marrow cultures the early expression of OPN is considerably higher (Yao et al., 1994). This difference appears to relate to the initial formation of a cernent layer in the mmw ce11 cultures that foms during the proliferative phase and which provides the substratum for subsequent bone nodule formation (Shen et al., 1997). In contrast, in the periosteal ceIl cultures the bone nodules form on a multilayer of flattened fibroblastic cells (Bhargava et al., 1988). Despite the low level of OPN mRNA expression in the proliferating periosteal cells, flow cytometry anaiysis revealed a high proportion of cells expressing intracellular OPN at all culture stages. At the early proliferative stage, confocal microscopy showed that the OPN+ve cells typically displayed a weak to moderate perinuclear staining or perimernbranous staining, whereas in matrix-fobg cultures stronger perinuclear staining was prominent. While al1 of the OPN+ve cells contributed to the amount of OPN measured within the cells, only the perinuclear staining is indicative of newly-synthesized OPN which should correlate with the mRNA expression. Thus, the higher than anticipated amount of OPN per ce11 and the high number of OPN+ve cells at the proliferative stage may reflect the presence of large numbers of OPN+ve migrant cells. Intracellular OPN content in FRCCs was related to the phase of the cell-cycle dependent and closely followed the general changes in ce11 protein content. Both OPN and the total protein content increased as the cells approached mitosis. The increase in OPN content as cells progressed through the ce11 cycle fkom Gi to rnitosis also indicates that OPN is not behaving as a nomal secreted protein (Ko et al., 1981). It is conceivable that cell populations with the perimembranous OPN may be responsible for this und relationship with the cell cycle. Moreover, the increase in the proportion of OPN+ve cells in populations approaching mitosis may relate to the expression of the non-secreted perimembranous OPN. Notably, CD44 and ePin appear to fom complexes with the intracellular OPN and are involved, not ody in ce11 migration as discussed detail below, but also may be involved in cell division (Tsukita and Yonemura, 1997). Thus, cytoskeletal intracellular OPN rnay be involved in the shape changes involved in cell division. Such a relationship could be deteminecl fiom immunocytochemical analyses of cells tmdergoing mitosis, as described previously for the ERM proteins (Tsukita and Yonernura, 1997).

Effects of Osteogenic Signals on lntraceIIular OPN The effects of dexarnethasone, TGF-B and BMP-7 (rhOP-l), representative hormones and cytokines with the capacity to influence bone ce11 differentiation, support the contention that OPN in combination with morphological cnteria can be used to characterize stages in osteogenic differentiation. Dexamethasone and TGF-P family members upregulate OPN expression in EXCC (Li et al., 1996; Nagata et al., 1991; Wrana et al., 1991). However, while dexarnethasone and rhOP- 1 induce osteogenic differentiation and bone nodule formation (Bellows et al., 1990) TGF-P 1, which increases matrix production and OPN expression, inhibits bone nodule formation (Wrana et al., 1991), even in the presence of rhOP-1 (Cheifetz et al., 1996). The flow cytometry analyses of htracelIu1a.r OPN expression showed that, while al1 of these agents upregulate intracellular OPN content, TGF-Pl treatment at pre- codiuence increases the proportion of OPN+ve cells and the amount of intracellular OPN, consistent with an increase in matrk production. In contrast, dexarnethasone and rhOP-1 induce a significantly higher percentage of OPN-ve celis which represent, as shown in Chapter-III. an early osteoprogenitor subpopulation. These data can explain the osteoinductive effects of dexamethasone and BMP-7. However, the osteoinductive effects of rhOP-1 appear to induce only pre-committed osteogenic subpopulations since &OP-1 promoted chondrogenic differentiation in less- differentiated cells and stem cells-

OPN-ve Cells 1 have identined OPN-ve subpopulations in primary cultures of the FRCC that were expanded at the proliferation and early mioeralization periods of the culture. This is the nrst study to isolate and characterize early precursor populations in bone lineage cultures that exhibit the classical criteria for stem cells (Hall and Watt, 1989; Potten and Loeffler, 1990). These criteria describe the stem ceiI as a nul1 cell: it lacks any propercies or features attributed to specialized cells. To characterize these cells I modified a flow cytometry system that had been used with Limited success in earlier studies (Turksen and Aubin, 1991; Van Vlasselaer et al., 1994). OPN-ve cells were identified as a population of small, quiescent celis with low cytoplamiic granularity. These physical characteristics facilitate the separation of subpopulations by the relatively atraumatic technique of flow cytometry. The latter feature was important for fiirther characterization of the sorted ce11 populations. Notably, the OPN-ve cells lacked expression of common differentiation markers such as type 1, II, and III collagens and alkaline phosphatase activity, and were deficient in CD44 receptors, which are expressed early in development (Toole, 1997). However, on plating these cells proliferate and begin expressing the aforernentioned differentiation markers, including OPN. This finding indicates the important role of ce11 attachent in promoting cellular differentiation. Despite the recognition of cells with stem ce11 characteristics within the S-ce11 population, it was difficult to ascertain the achial proportion of cells that were functional, mesenchymal stem cells. The basic problem is the lack of a dennitive rnarker for mesenchymal stem cells and as a consequence analyses are dependent upon the differentiation of the stem cells into a recognizable phenotype that cm be rnonitored. Thus, when analyzed by osteogenic differentiation ody 0.5% of the cells formed bone nodules. However, osteogenic precursor/transit cells cari also generate bone nodules, albeit of smaller size, as evident in cultures of L-cell and S-ce11 populations. While this would indicate that the proportion of stem cells is less than OS%, stem cells generate other lineages which must be included in their total differentiation repertoire. Thus, fiom studies in which the sorted ceus were plated at low density it was evident thaf while osteogenic differentiation was prominent, differentiation dong chondrogenic, adipocytic, and fibrogenic pathways also occured. In addition, while the viability of the S cell population was only slightly less than unfiractionated cells, the viability of the stem cells within the population is not known. Other factors, such as the culture environment including the substratum and culture medium can aiso affect the viability and ability of the stem ceiis to differentiate. There is also indirect evidence to indicate that stem cells are sensitive to their cornpartment size and to the presence of differentiated progeny in their surroundings (Potten and Loeffler, 1990). Consequently, the precise nurnber of stem celis can not be determined until specific markers cm be identified. However, the proposed in vivo relationship between the quiescent stem ce11 hction and the transit amplifying cells could be examined by administering high specific activity 'H- thymidine (McCulloch et al, 1991) at dinerent intervals starting kom sorted OPN-ve cells in suspension to cells at day 3 after plating. Incorporation of isotope into cycling cells causes ce11 death, and based on the production of bone cartilage, fat and collagen, the intervals at wvhich the different phenotypes were detected would give an estimate of the times when the stem cells and amplimg cells were proli ferating. Although stem cells fiom fetal periosteal tissues generated multiple phenotypes, differentiation dong an osteogenic pathway was favoured. Currently, it is UnkIlown whether this reflects differences in the relative proportions of osteoprogenitor ce11 populations within specific stromd tissues or whether culture conditions bias the differentiation process. Experiments in which comparable populations are obtained from other fetd strornal (e-g. bone marrow) tissues and analyzed for their differentiation potential could provide insights into this question. In addition, the influence of substratum on strornal ce11 differentiation could be analyzed by coating culture dishes with various extracellular matrix macrornolecules such as collagen, fibronectin, laminin, or bone ma& proteins, prior to low density plating of S-ce11 populations. In this respect the effects of hyaluronan, which is abundant in fetal tissues and in sites of wound healing and tissue regeneration (Cabrera et al., 1995; Gerdin and Hallgren, 1997). would be particularly interesting. Sirnilarly the effects of hormones and cytokines could be assessed under dehed substratum conditions. Although the interpretation of data could be compromised to some degree by the presence of fetal bovine serum, which in itself creates a wound-like environment for the cells, the serum did not block the abiiity of rhOP-1 to promote chondrogenic differentiation in the S-ce11 popdation. Nevertheless, Mer enrichment or isolation of stem celis would facilitate the interpretation of experiments such as these, Wer stressing the importance for identifying markers for stem cells. An ability to isolate and expand stem cells in culture has fundamental importance in reconstnictive surgery. The restricted ability of higher vertebrates to repair and regenerate lost tissues and organs has been suggested to reflect an age- dependent decrease in stromal stem cells and progeniton (Owen, 1985). Miile the hi@ proliferative capacity and multipotentiality of stem cells is an important response to noxious stimuli and damage to tissues, the ability to regenerate lost tissues requires that an appropriate environment and a sufficient number of stem cells are available to recapitulate the developrnental processes required to re-structure lost tissues. Although the property of self-renewal including asymmetnc mitosis is thought to provide a reservoir of stem cells with parental strand DNA and therefore reduced likelihood of genomic DNA infidelity (Potten, 1976), declining nurnbers of these cells could compromise regenerative processes. Thus, the introduction of exogenous stem cells into healing tissues has considerable potential as an adjunct to surgical intervention. While my studies have demonstrated the feasibility of isolating viable populations of cells enriched with stromai stem cells it will be important to develop simpler and less expensive procedures for potential clinical applications. Studies of haematopoietic stem cells have identified ce11 surface marken, such as Sca- 1 and CD34 that have been used to isolate stem cells (Allen et al., 1984; Taichman et al., 1996; Verbik et al., 1995). The ability to obtain emiched stromal stem ce11 populations should facilitate the identification of cell-surface markers using monoclonal antibody, peptide display and single-ceIl SAGE analyses. Monoclonal antiiodies can be generated against whole ceils and specific monoclonals expanded and used to isolate subpopulations in a viabIe foxm by immunopaxhg. SSunilarly peptide display, which has been used to identify specific ceU-surface receptors in endothelial cells forming the microvasculature of different tissues (Ruoslahti, 1997) can be used to selectively bind subpopulations of ceUs for isolation and Mercharacterization. Applying the SAGE technique to mRNA isolated fkom single ceils can generate ESTs that characterize the cell phenotype and can identify unique gene products that cm be used for isolation and characterization.

OPN ni Migrating Ceiis The observation that OPN is present within discrete foci with a perimembranous distribution and which are prominent in migrating cells provided the impetus for more detailed studies to characterize this intracellular form of OPN. The high levels of intracelMar OPN content in fetal rat migrating cells shown in Chapter II (Zohar et al., 1997a) were analyzed using confocal microscopy which revealed a predominance of subcortical OPN accumulating in lamellipodia-like extensions, consistent with an increased expression of perimembranous OPN in migrating cells. Because of the unwual location of the OPN, a series of different antibodies was used to CO- these findings. Studies were extended to cells derived from different tissues and species to demonstrate that this was a general phenomenon for cells with embryonic characteristics. Since extracellular OPN had been shown to associate with the hyaluronan nansmembrane receptor CD44 (Weber et al., 1997) which is characteristically expressed in embryonic tissues, the relationship between the perimembranous OPN, CD44 and hyaluronan was determined by confocal microscopy and extended to include ERM proteins. Not only did these experiments indicate a unique involvement of OPN in the CD44 attachent complex in embryonic cells, but a sirnilar relationship was revealed in tumour cells and activated macrophages, which are also characterized by migratory activity in metastasis and extravasation, respectively. The upregulation of OPN and CD44 in metastatic cells (Senger et al., 1979; Weber et al., 1997) and activateci lymphocytes and macrophages (Patarca et al., 1993) is also consistent with the relationship of these proteins in an attachent cornplex.

The association of OPN with the hyaluronan-CD44 complex was also indicated fiom Western blot analysis of components isolated hm hyaluronan-bead extractions which revealed an enrichment for CD44 OPN and ERM proteins. Although OPN has been shown to bind CD44, extracellular binding of OPN and HA to CD44 are mutually exclusive (Weber et al., 1997). Therefore, the enrichment of OPN in complexes isolated f?om hyaluronan-beads is not Likely to be extracellular, consistent with the immunocytochemical staining which revealed perimembranous OPN only after permeabilization of the cells. The intracelluiar association of OPN with CD44 is also supported by the lack of biotinylated OPN in CD44 complexes isolated after ceil-dace biotinylation. Although it is conceivable that OPN could be phagocytosed or endocytosed, no evidence of internalization of exogenous OPN was evident. If the perimembranous OPN remains inside the ce11 following synthesis, the existence of an mRNA transcnpt lacking the leadhg sequence might be anticipated. Indeed, alternative splicing in the 5'-UTR of OPN has been reported (Singh et al., 1992) but in this particular transcript the leader sequence was retained. The presence of mRNA lacking the leader sequence can be determined using PCR with primers matching sequences in exons flanking the exon that codes for the leader sequence and analyzing for truncated amplicons.

Although the existence of an intracellular form of a secreted protein is unusual, the intracellular association of calreticulin with the cytoplasrnic domains of integrin a- subunits provides an interesthg analogy. Calreticulin was origulally characterized as an ER protein that functiow as a molecular chaperone (Waser et al., 1997). However, a binding motif in the calreticulin sequence led to its demonstration in integrin complexes (Leung-Hagesteijn et al., 1994) and its ability to inhibit nuclear steroid hormone receptors (Dedhar, 1994). While its presence in the nucleus is controversial, a functional role for calreticulin in the adhesion and signalling properties of integrin complexes has been established (Coppolino et al., 1997). Notably like OPN, caireticuiin cm be phosphorylated and is a calcium binding protein (Waser et al., 1997). 152 Based on the CO-localization of CD44 OPN and ERM proteins and the hyaluronan, present on coated beads, a model of the HA-CD44-ERM complex incorporating the OPN is shown in Fig VI. 1. The evidence that support this mode1 is as follows: First, it has been established that CD44 is a receptor for hyaluronan and extracellular OPN but that the two ligands have differentiatial effects. Whereas hyaluronan promotes cellular aggregation (Maieski and Knudson, 1996), OPN encourages chemotactic activity (Weber et al., 1996). However, both hyaluronan and OPN appear to be involved in cell motiiity. CD44 has already been shown to associate with the ERM proteins (Tsukita et al., 1994), an interaction that could be inhibited by antibodies to the cytoplasmic domain of CD44 (Hirao et al., 1996). In the model shown in Fig VI. 1, 1 propose that a 72 kDa intracellular protein show to be involved in the association of CD44 and ERM (Tsukita et al., 1994) is intracellular OPN, based on the data presented in Chapters II & V. Consequently, OPN may provide the link between CD44 and proteins that bind to the actin cytoskeleton. hterestingly, the up-regulation of OPN, CD44 and ERM proteins observed in metastatic cells is also observed with EGF induction (Chackalaparampil et al., 1996; Kneg and Hunter, 1992), the over-expression of which has been directly linked with ce11 transformation. Moreover, the tyrosine kinase activity of the EGF receptor and Rac and Rho proteins can convert inactive forms of ERMs to active CD44-binding forms (Mackay et al., 1997) and EGF also stimulates PI3 kinase activity which promotes actin assembly by abrogating gelsolin binding to actin filaments.

The link between CD44 and ERM is controlled by various phosphorylations of the ERM and CD44 These phosphorylations are mediated through tyrosine kinases associated with the EGF receptor (Kneg and Hunter, 1992) or serine/threonine phosphorylation shown to be involved in ERM activation (Nakamura et al., 1995). Fuaher, the capacity of CD44 to bind hyaluronan is dependent on phosphorylation of its cytoplasmic domain (Pure et al., 1995). Since OPN has concensus sites for both ser/thr and tyr phosphorylation (Denhardt and Guo, 1993) and recombinant OPN has autokinase activity (Ashkar et al., 1993), phosphorylation reactions may also control the association of OPN with this complex. Additionaiiy, ERM and CD44 can associate in the presence of phosphoinositides such as phosphatidylinositol Cmonophosphate (Hirao et al., 1996), while OPN in ceil lysates dlalso stimulate phosphatidylinositol 3- hydroxyl kinase (PU-kinase) activity (Chellaiah and Hruska, 1996). Although this has been associated with integrin binding it is conceivable that a similar activity might be associated with the intracelluiar OPN. Also, of relevance is the ability of OPN to chelate calcium ions which could influence calcium signalling transients inside the ce11 and regdate the binding of gelsolin to actin and modulate its severing activity (Janmey et al., 1990; Stossel et al., 1985).

Although a direct association of the CD44 complex with actin would be anticipated in view of its relationship to ceii mobility, in calcium-f?ee buffer actin was noticeably absent from complexes imrnunoprecipitated with CD44 antibodies îÎom hyaluronan-bead extracts. Also, confocal microscopy failed to show any definitive spatial association of intracellular OPN with actin filaments stained with phalloidin. Additionally, another abundant focal adhesion protein, vinculin did not exhibit e~chrnentin the CD44 immunoprecipitation or specific codistribution with OPN by irnmunostaining. Other studies have indicated that ezrin is not associated with actin filaments under physiological conditions (Shuster and Hennan, 199 5). Taken together these results support an earlier suggestion that ERM are not associated with classical focal adhesion complexes (Franck et al., 1993) and therefore, the CD44-ERM-OPN complex associated with hyaluronan appears to be a novel adhesion complex, perhaps similar to the close contacts identified by Abercombie (1976). The enhanced motility of cells with increased levels of intracellular OPN and the reduced motility of cells without OPN indicates that the intracellular OPN in the CD44-ERM-OPNcomplex is required for rapid ce11 migration while the classical focal adhesions of cultured cells are enriched with actin filaments and vinculin. These attachments are associated with well spread, slowly moving cells. In contrast, the adhesions to hyaluronan identified in my thesis are not apparently actin-bound; these types of more Beeting, evanescent contacts may facilitate rapid ce11 migration in embryonic or wound healing enwonments emiched in hyduronan. The identification of an actin-independent adhesion complex is paaicularly novel in view of current concepts of cell migration which emphasize actin treadmilling. Thus, the CD44-ERM-OPN complex may provide a stabilizing fiinction and not a tractional role.

It has been suggested that CD44 binding by hyaluronan does not involve signal transduction (Entwistle et al., 1996; Koshiishi et al., 1994) since there is no report of correlation between CD44 and intracellular signaling pathway. However, the relationship between CD44 expression and induction of processes such as wound healing (Alaish et al., 1994), -or development (Bartolazzi et al., 1994), and changes in cell motility (Kaaijk et al., 1995) can not be interpreted only by the ability of cells to adhere to hyaluronan. To determine whether signalling can occur through this complex following ligation of the CD44 by hyaluronan, or extracellular OPN, the ability of nomal fetd dermal cells to increase intracellular calcium or to phosphorylate components of the attachent complex could be determined immediately following attachment of the cells to hyaluronan- or OPN-coated substrata. The specificity for this complex and the involvement of OPN cm be mer evaluated by repeating these experiments with OPN-/-ve and CD44-/-ve cells.

The existence of the hyaluronan-CD44 complex appears to be restricted to rapidly migrating fetal cells, but cm be induced by EGF (Chackalaparampil et al., 1996; Krieg and Hunter, 1992) and the complex is present in activated immune cells (Patarca et al., 1993) and metastatic cells (Senger et al., 1979). Notably, ligation of CD44 receptors by HA promotes tumor development, progression and metastasis (Bartolazzi et al., 1994). Metastatic and immune cells display fast migration in matrices enriched with hyaluronan consistent with the notion that these cellular matrix contacts are induced to enable rapid motility of these cells.

Further support for the involvement of an intracellular OPN in this complex will require a more definitive charactenzation of the OPN and its ability to bind to various components in the complex. Two approaches can be utilized to isolate the intracellular OPN nom secreted OPN. Since migrant cells predorninantly express the intracellular perimembranous OPN, separation of migrant cells on a preparative Boyden chamber system, as described in Chapter V, could enrich for cells with the intracellular OPN which can then be isolated wing antibody afnnity columns and ion-exchange 155 chromatography. A second approach is to pulse label ceus and follow this with a 3h chase the to aUow essentially alI of the labeled, secreted OPN to leave the cell. However, as the half-life of the intracelldar OPN is unknown preliminary experiments would be needed to detennine optimal labehg conditions. The properties of the radiolabeled intracellular OPN (including post-translational modifications) cm be compared with secreted OPN. Notably, combining the two approacha could f.urther select for the intracellula. OPN. Of particular interest in the characterization of the intracellular OPN would be its ability to bind to the cytoplasmic domain of CD44 and to ERM proteins, which can be analyzed by Western blotting.

OPN-nul1 Mice Although generation of transgenic animals has been usefid in establishing the hction of specific proteins by selectively inactivating genes, in many instances a clea. difference in phenotype is not observed. This has generally been the situation for bone matrix proteins including osteocalcin @ucy et al., 1996), bone sialoprotein (Aubin et al., 1996) and also OPN (Liaw and Hogan, 1996; Rittling et al., 1997). In such cases it is believed that there is a fùnctional redundancy or that compensatory rnechanisms exist. The nomal phenotype of OPN-nul1 mice indicates that OPN expression rnay not be essential for development or for critical functions in the adult animal. Surprisingly, no apparent defects have been observed in bone where OPN is especially abundant in cernent layers. However, it is possible that more subtle effects may become apparent on more detailed examination of these animals. Thus, it is evident that while the OPN-/-ve cells derived £iom OPN-nul1 rnice cells attach to collagen theu ability to attach to hyaluronan and rnigrate are markedly irnpaired. Since the intracellular OPN that is associated with CD44 is present only in cells with embryonic charactenstics this impairment would be evident only in fetal and reparative tissues. Without totally blocking these hincîions it is possible that developmental and regenerative processes may not be af3ected when examined macroscopically. Thus, it will be of interest to study developmental processes in these mice as well as their wound heaiing and immune response capabilities. Additionally, examination of the metastatic potential of tumours forrned in these mice could provide insights into the function of the intraceilular OPN.

Sammary & Conclusions

The principle conclusions obtained from my studies of intracellular OPN expression are as follows: A combination of rnorphological features and intracellular OPN expression cm define different stages of bone ce11 differentiation. Two distinct phenotypes cm be recognized in FRCCs (based on OPN immunostaining) that may represent discrete cell populations. Perimembranous intracellula. OPN is enricheci in fetal migrating stromal cells. The smd, quiescent, OPN-ve cek present at various stages of osteogenesis in FRCC cultures can be isolated by flow cytometry as a viable subpopulation that is emiched for multipotential stem ceiIs and for osteoprogenitor ceiis The stem cells in FRCCs have been characterized as quiescent cells of small size, low grand&, high nuclea.to cytoplasmic ratio. They exhibit pluripotentiality, high prolifdve capacity and capacity for self-renewal, but do not express differentiaîion-associated markers (collagen types 1, 4 III, alkaline phosphatase, osteopontin, CD44). FRCC cm support the growth and differentiation of hernatopoietic ceIls, even without exogenous administration of hernatopoietic ce11 growth factors (Le. erythropoietin, SCF, IL-3, iL-6). rhOP-1 promotes osteogenic differentiation of cells already committed to the osteogenic heage whereas undifferentiated cells are induced to differentiate dong the chondrogenic pathway. The intracellular form of OPN is an integrai component of a hyaluronan-CD44 ERM attachment cornplex that is involved in the migration of fetal and metastatic cells, and activated macrophages. The hy aluronan adhesion complexes require intracellular OPN expression for motility function but are not associated directly with actin or vinculin. Collectively, these results have shown that expression of intracellular OPN alone is hadequate for discriminating stages of osteogenïc differentiation. However, when used in combination with morphological criteria OPN can be useful for studies of osteogenesis, as supported by the effects of homones and cytokines that modulate OPN expression in concert with osteogenic differentiation. However, of more significance has been the identification and characterization of stromal stem cells, which were initially recognized in populations of fetd ceils that did not express OPN. These studies have provided the £kt direct evidence for the existence of stem cells in stromal ce11 populations. The limitations of OPN as an osteogenic marker is primarily due to the expression of a unique intracellular fom of OPN, which 1 have shown to be associated with the HA-CD44-ERM cell attachment complex involved in ceil motility. The involvement of OPN in this complex has far-reaching implications in the motility of cells during development and regeneration, in the immune response and in the metastasis of cancer cells. 1 Motility 1

Actin

Fig. VI. 1- Mode1 Depicted The Association of The Intracellular OPN to the HyaIumoan-CD44-ERM Adhesion Complexes. CHAPTER VU

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